Methods and compositions for biomarkers of fatigue

ABSTRACT

The present invention provides methods and compositions for identifying fatigue, disease states associated with fatigue, recovery from fatigue and/or physical performance capability in a subject.

STATEMENT OF PRIORITY

This application is a continuation application of, and claims priorityto, U.S. application Ser. No. 13/839,332, filed Mar. 15, 2013, which isa continuation-in-part application of, and claims priority to, PCTApplication No. PCT/US2012/064798, filed Nov. 13, 2012, which claims thebenefit, under 35 U.S.C. §119(e), of U.S. Provisional Application Ser.No. 61/559,632, filed Nov. 14, 2011, the entire contents of each ofwhich are incorporated by reference herein.

STATEMENT REGARDING ELECTRONIC FILING OF A SEQUENCE LISTING

A Sequence Listing in ASCII text format, submitted under 37 C.F.R.§1.821, entitled 9556-4IP_ST25.txt, 187,949 bytes in size, generated onSep. 30, 2013 and filed electronically via EFS-Web, is provided in lieuof a paper copy. This Sequence Listing is hereby incorporated byreference into the specification for its disclosures.

FIELD OF THE INVENTION

The present invention relates to biomarkers and methods of their use inidentifying fatigue, disease states associated with fatigue, recoveryfrom fatigue and/or physical performance capability in a subject.

BACKGROUND OF THE INVENTION

There is great interest in finding methods that can be used todiagnosis, evaluate and/or monitor objectively the disease statereferred to as chronic fatigue syndrome (CFS). As one example, anobjective, saliva-based measurement tool would be useful in determiningwhether an individual is experiencing a level of fatigue sufficient tomeet a diagnosis of chronic fatigue syndrome. Other applications includemonitoring changes in fatigue level, e.g., in a subject diagnosed withCFS.

The present invention provides methods and compositions for diagnosingCFS, identifying subjects having an increased risk or likelihood ofhaving or developing CFS and/or monitoring or evaluating fatigue in asubject by detecting and/or measuring biomarkers in one or more samplesfrom the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. For the biomarker bm_cfs_cand3, levels are greater in salivafrom individuals with chronic fatigue syndrome (CFS) than in saliva fromnon-fatigued, control individuals (NF). The base 10 logarithm of the ionintensity, as determined by mass spectrometry, is shown as a function ofpatient type, i.e., CFS vs. NF. Data are shown as boxplots with thesolid black line indicating the group median. The hollow box around thesolid black line indicates the bounds of the data from the first to thethird quartile. The whiskers indicate a distance 1.5× greater than theinterquartile range from the nearest edge of the box. A non-parametrictest suggested the two samples were unlikely to arise from a commondistribution (Wilcoxon rank sum test, p<0.05).

FIG. 2. For the biomarker bm_cfs_cand4, levels are greater in salivafrom individuals with chronic fatigue syndrome (CFS) than in saliva fromnon-fatigued, control individuals (NF). The base 10 logarithm of the ionintensity, as determined by mass spectrometry, is shown as a function ofpatient type, i.e., CFS vs. NF. Data are shown as boxplots with thesolid black line indicating the group median. The hollow box around thesolid black line indicates the bounds of the data from the first to thethird quartile. The whiskers indicate a distance 1.5× greater than theinterquartile range from the nearest edge of the box. A non-parametrictest suggested the two samples were unlikely to arise from a commondistribution (Wilcoxon rank sum test, p<0.05).

FIG. 3. For the biomarker sp_6, levels are greater in saliva fromindividuals with chronic fatigue syndrome (CFS) than in saliva fromnon-fatigued, control individuals (NF). The base 10 logarithm of the ionintensity, as determined by mass spectrometry, is shown as a function ofpatient type, i.e., CFS vs. NF. Data are shown as boxplots with thesolid black line indicating the group median. The hollow box around thesolid black line indicates the bounds of the data from the first to thethird quartile. The whiskers indicate a distance 1.5× greater than theinterquartile range from the nearest edge of the box. A non-parametrictest suggested the two samples were unlikely to arise from a commondistribution (Wilcoxon rank sum test, p<0.05).

FIG. 4. A flow chart regarding the inclusion/exclusion of patients,showing procedure for obtaining 46 and 45 CFS and control salivasamples, respectively, from 21,165 subjects.

FIG. 5. CFS biomarker levels in CFS and normal subjects. Y-axis showsthousands of intensity units corresponding to concentration of CFSbiomarker peptide in saliva normalized to total protein. Each circlerepresents a single sample.

FIG. 6. ROC curve for CFS salivary biomarker. Heavy solid line indicatessensitivity as a function of specificity (100-sensitivity). Dotted linesindicate 95% confidence interval. Light diagonal line indicatesrelationship between sensitivity and specificity if both CFS and controlpopulations are the same. P-value associated with a comparison betweenthe observed ROC AUC and ROC AUC=0.5 (no discrimination between diseasedand normal) is <0.0001.

SUMMARY OF THE INVENTION

In some embodiments, the present invention provides a method of guidinga human subject's sleep schedule, comprising: a) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject; b)calculating the ratio of the concentration of the peptide(s) measured in(a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) having the subject initiate or resume a sleep schedule;d) measuring the concentration of a peptide selected from the groupconsisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject at one or more time points after (c), wherein thepeptides of (d) are the same as the peptides of (a); e) calculating theratio of the concentration of the peptide(s) measured in (d) to thetotal amount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) guiding the subject's sleep schedule by modifyingthe duration of subsequent sleep periods using the subject's ratio(s) ascalculated in (e), such that an increase in the ratio relative to theprevious ratio leads to a subsequent increase in the duration of thesubject's sleep period, and a decrease in the ratio or a constant ratiorelative to the previous ratio leads to no change in the duration of thesubject's sleep period or a subsequent decrease in the duration of thesubject's sleep period.

In further embodiments, the present invention provides a method ofguiding a human subject's use of a sleep enhancing material and/or sleepenhancing activity, comprising: a) measuring the concentration of apeptide selected from the group consisting of: 1) a peptide comprisingthe amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) apeptide comprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ(SEQ ID NO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from the subject; b) calculating theratio of the concentration of the peptide(s) measured in (a) to thetotal amount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) exposing the subject to the sleep enhancing materialand/or sleep enhancing activity; d) measuring the concentration of apeptide selected from the group consisting of: 1) a peptide comprisingthe amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) apeptide comprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ(SEQ ID NO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from the subject at a time point afterstep (c), wherein the peptides of (d) are the same as the peptides of(a); e) calculating the ratio of the concentration of the peptide(s)measured in (d) to the total amount of protein in the sample, accordingto the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) guiding the subject's use of the sleep enhancingmaterial and/or sleep enhancing activity using the subject's ratio(s) ascalculated in (e), such that an increase in the ratio relative to theprevious ratio leads to a subsequent increase in the subject's use ofthe sleep enhancing material and/or sleep enhancing activity, and adecrease in the ratio or a constant ratio relative to the previous ratioleads to no change or a subsequent decrease in the subject's use of thesleep enhancing material and/or sleep enhancing activity.

The present invention additionally provides a method of guiding a humansubject's treatment of a sleep disorder, comprising: a) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject; b)calculating the ratio of the concentration of the peptide(s) measured in(a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) treating the subject for the sleep disorder; d)measuring the concentration of a peptide selected from the groupconsisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject at a time point after step (c), wherein the peptides of(d) are the same as the peptides of (a); e) calculating the ratio of theconcentration of the peptide(s) measured in (d) to the total amount ofprotein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) guiding the subject's treatment of the sleepdisorder using the subject's ratio(s) as calculated in (e), such that anincrease in the ratio relative to the previous ratio leads to asubsequent enhancement of the treatment for the sleep disorder, and adecrease in the ratio or a constant ratio relative to the previous ratioleads to no change or a subsequent reduction of the treatment for thesleep disorder.

Further provided herein is a method of identifying a substance and/oractivity that enhances sleep, comprising: a) measuring the concentrationof a peptide selected from the group consisting of: 1) a peptidecomprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ IDNO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject; b)calculating the ratio of the concentration of the peptide(s) measured in(a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) exposing the subject to the test substance and/or testactivity; d) measuring the concentration of a peptide selected from thegroup consisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject at a time point after step (c), wherein the peptides of(d) are the same as the peptides of (a); e) calculating the ratio of theconcentration of the peptide(s) measured in (d) to the total amount ofprotein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) determining if the test substance and/or testactivity enhances sleep using the subject's ratio(s) as calculated in(e), such that a decrease in the ratio relative to the previous ratioidentifies the test substance and/or test activity as a substance and/oran activity that enhances sleep.

In addition, the present invention provides a method of identifying asubstance and/or activity that treats a sleep disorder, comprising: a)measuring the concentration of a peptide selected from the groupconsisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject; b) calculating the ratio of the concentration of thepeptide(s) measured in (a) to the total amount of protein in the sample,according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) exposing the subject to the test substance and/or testactivity; d) measuring the concentration of a peptide selected from thegroup consisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject at a time point after step (c), wherein the peptides of(d) are the same as the peptides of (a); e) calculating the ratio of theconcentration of the peptide(s) measured in (d) to the total amount ofprotein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) determining if the test substance and/or testactivity treats the sleep disorder using the subject's ratio(s) ascalculated in (e), such that a decrease in the ratio relative to theprevious ratio identifies the test substance and/or test activity as asubstance and/or an activity that treats the sleep disorder.

A method is also provided herein of identifying a human subject that issleep deprived, comprising: a) measuring the concentration of a peptideselected from the group consisting of: 1) a peptide comprising the aminoacid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ IDNO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from each subject in a population ofsubjects that are not sleep deprived; b) calculating the ratio of theconcentration of the peptide(s) measured in (a) to the total amount ofprotein in each sample of (a), according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for each of the study subjects in thepopulation of (a); c) establishing a threshold ratio for the populationof subjects of (a); d) measuring the concentration of a peptide selectedfrom the group consisting of: 1) a peptide comprising the amino acidsequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ IDNO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from a test subject; e) calculatingthe ratio of the concentration of the peptide(s) measured in (d) to thetotal amount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for the test subject; and f) comparingthe ratio of the test subject with the threshold ratio of (c), whereby aratio of the test subject that is greater than the threshold ratio of(c) identifies the test subject as being sleep deprived.

Also provided herein is a method of identifying a human subject that issleep deprived, comprising: a) measuring the concentration of a peptideselected from the group consisting of: 1) a peptide comprising the aminoacid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ IDNO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from the subject; b) calculating theratio of the concentration of the peptide(s) measured in (a) to thetotal amount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for the subject; and c) comparing theratio of the subject with a threshold ratio, whereby a ratio of thesubject that is greater than the threshold ratio identifies the subjectas sleep deprived.

In additional embodiments, the present invention provides a method ofguiding a human subject's treatment of fatigue, comprising: a) measuringthe concentration of a peptide selected from the group consisting of: 1)a peptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject; b)calculating the ratio of the concentration of the peptide(s) measured in(a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) treating the subject for fatigue; d) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject at atime point after step (c), wherein the peptides of (d) are the same asthe peptides of (a); e) calculating the ratio of the concentration ofthe peptide(s) measured in (d) to the total amount of protein in thesample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) guiding the subject's treatment of fatigue usingthe subject's ratio(s) as calculated in (e), such that a an increase inthe ratio relative to the previous ratio leads to a subsequentenhancement of the treatment for fatigue, and a decrease in the ratio ora constant ratio relative to the previous ratio leads to no change or asubsequent reduction of the treatment for fatigue.

Further provided herein is a method of identifying a substance and/oractivity that reduces fatigue, comprising: a) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject; b)calculating the ratio of the concentration of the peptide(s) measured in(a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) exposing the subject to the test substance and/or testactivity; d) measuring the concentration of a peptide selected from thegroup consisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject at a time point after step (c), wherein the peptides of(d) are the same as the peptides of (a); e) calculating the ratio of theconcentration of the peptide(s) measured in (d) to the total amount ofprotein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) determining if the test substance and/or testactivity reduces fatigue using the subject's ratio(s) as calculated in(e), such that a decrease in the ratio relative to the previous ratioidentifies the test substance and/or test activity as a substance and/oran activity that reduces fatigue.

Additionally provided herein is a method of guiding a human subject'streatment of a chronic fatigue syndrome (CFS), comprising: a) measuringthe concentration of a peptide selected from the group consisting of: 1)a peptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject; b)calculating the ratio of the concentration of the peptide(s) measured in(a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) treating the subject for CFS; d) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject at atime point after step (c), wherein the peptides of (d) are the same asthe peptides of (a); e) calculating the ratio of the concentration ofthe peptide(s) measured in (d) to the total amount of protein in thesample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) guiding the subject's treatment of CFS using thesubject's ratio(s) as calculated in (e), such that an increase in theratio relative to the previous ratio leads to a subsequent enhancementof the treatment for CFS, and a decrease in the ratio or a constantratio relative to the previous ratio leads to no change or a subsequentreduction of the treatment for CFS.

The present invention also provides a method of identifying a substanceand/or activity that treats chronic fatigue syndrome (CFS), comprising:a) measuring the concentration of a peptide selected from the groupconsisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject; b) calculating the ratio of the concentration of thepeptide(s) measured in (a) to the total amount of protein in the sample,according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) exposing the subject to the test substance and/or testactivity; d) measuring the concentration of a peptide selected from thegroup consisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject at a time point after step (c), wherein the peptides of(d) are the same as the peptides of (a); e) calculating the ratio of theconcentration of the peptide(s) measured in (d) to the total amount ofprotein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) determining if the test substance and/or testactivity treats CFS using the subject's ratio(s) as calculated in (e),such that a decrease in the ratio relative to the previous ratioidentifies the test substance and/or test activity as a substance and/oran activity that treats CFS.

Additionally provided herein is a method of identifying a human subjecthaving an increased likelihood of having or developing chronic fatiguesyndrome (CFS), comprising: a) measuring the concentration of a peptideselected from the group consisting of: 1) a peptide comprising the aminoacid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ IDNO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from each subject in a population ofsubjects that do not have a diagnosis of CFS or symptoms of CFS; b)calculating the ratio of the concentration of the peptide(s) measured in(a) to the total amount of protein in each sample of (a), according tothe equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for each of the study subjects in thepopulation of (a); c) establishing a threshold ratio for the populationof subjects of (a); d) measuring the concentration of a peptide selectedfrom the group consisting of: 1) a peptide comprising the amino acidsequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ IDNO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from a test subject; e) calculatingthe ratio of the concentration of the peptide(s) measured in (d) to thetotal amount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for the test subject; and f) comparingthe ratio of the test subject with the threshold ratio of (c), whereby aratio of the test subject that is greater than the threshold ratio of(c) identifies the subject as having an increased likelihood of havingor developing CFS.

In further aspects of this invention, a method is provided ofidentifying a human subject having an increased likelihood of having ordeveloping chronic fatigue syndrome (CFS), comprising: a) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject; b)calculating the ratio of the concentration of the peptide(s) measured in(a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for the subject; and c) comparing theratio of the subject with a threshold ratio, whereby a ratio of thesubject that is greater than the threshold ratio identifies the subjectas having an increased likelihood of having or developing CFS.

Additionally provided herein is a method of guiding a human subject'swork load, comprising: a) measuring the concentration of a peptideselected from the group consisting of: 1) a peptide comprising the aminoacid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ IDNO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from the subject; b) calculating theratio of the concentration of the peptide(s) measured in (a) to thetotal amount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) having the subject initiate or resume a work load; d)measuring the concentration of a peptide selected from the groupconsisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject at one or more time points after (c), wherein thepeptides of (d) are the same as the peptides of (a); e) calculating theratio of the concentration of the peptide(s) measured in (d) to thetotal amount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) guiding the subject's work load by modifying theduration of the subject's work period and/or amount of work the subjectdoes using the subject's ratio(s) as calculated in (e), such that anincrease in the ratio relative to the previous ratio leads to asubsequent decrease in the duration of the subject's work period and/ora decrease in the amount of work the subject does, and a decrease in theratio or a constant ratio relative to the previous ratio leads to nochange in the duration of the subject's work period and/or amount ofwork the subject does or a subsequent increase in the duration of thesubject's work period and/or amount of work the subject does.

In further aspects, the present invention provides a method ofidentifying a human subject sufficiently rested to carry out a workload, comprising: a) measuring the concentration of a peptide selectedfrom the group consisting of: 1) a peptide comprising the amino acidsequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ IDNO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from each subject in a population ofsubjects that have carried out the work load sufficiently, wherein thesample is taken from each subject at about the time the subject startsthe work load; b) calculating the ratio of the concentration of thepeptide(s) measured in (a) to the total amount of protein in the sample,according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for each of subject in the populationof subjects of (a); c) establishing a threshold ratio for the populationof subjects of (a); d) measuring the concentration of a peptide selectedfrom the group consisting of: 1) a peptide comprising the amino acidsequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ IDNO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from a test subject; e) calculatingthe ratio of the concentration of the peptide(s) measured in (a) to thetotal amount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for the test subject; and f) comparingthe ratio of the test subject with the threshold ratio of (c), whereby aratio of the test subject that is less than or equal to the thresholdratio of (c) identifies the test subject as being sufficiently rested tocarry out the work load and a ratio of the test subject that is greaterthan the threshold ratio of (c) identifies the test subject as not beingsufficiently rested to carry out the workload.

Additionally provided herein is a method of identifying a human subjectthat is sufficiently rested to carry out a work load, comprising: a)measuring the concentration of a peptide selected from the groupconsisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject; b) calculating the ratio of the concentration of thepeptide(s) measured in (a) to the total amount of protein in the sample,according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for the subject; and c) comparing theratio of the subject with a threshold ratio, whereby a ratio of thesubject that is less than or equal to the threshold ratio identifies thesubject as being sufficiently rested to carry out the work load and aratio of the subject that is greater than the threshold ratio identifiesthe subject as not being sufficiently rested to carry out the work load.

Additionally, the present invention provides a method of identifying ahuman subject who is fit for duty, comprising: a) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from each subject in apopulation of subjects that are fit for duty: b) calculating the ratioof the concentration of the peptide(s) measured in (a) to the totalamount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for each subject in the population ofsubjects of (a); c) establishing a threshold ratio for the population ofsubjects of (a); d) measuring the concentration of a peptide selectedfrom the group consisting of: 1) a peptide comprising the amino acidsequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ IDNO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from a test subject; e) calculatingthe ratio of the concentration of the peptide(s) measured in (d) to thetotal amount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for the test subject; and f) comparingthe ratio of the test subject with the threshold ratio of (c), whereby aratio of the test subject that is greater than the threshold ratio of(c) identifies the test subject as being not fit for duty, and a ratioof the test subject that is the same as or less than the threshold ratioof (c) identifies the test subject as being fit for duty.

Furthermore, the present invention provides a method of identifying ahuman subject who is fit for duty, comprising: a) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject; b)calculating the ratio of the concentration of the peptide(s) measured in(a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg) to determine a ratio for the subject; and c) comparing theratio of the subject with a threshold ratio, whereby a ratio of thesubject that is greater than the threshold ratio identifies the subjectas being not fit for duty, and a ratio of the subject that is the sameas or less than the threshold ratio identifies the subject as being fitfor duty.

In a further aspect, the present invention provides a method ofidentifying a subject as having chronic fatigue syndrome or having anincreased likelihood of having or developing chronic fatigue syndrome,comprising: a) measuring the amount of a peptide comprising the aminoacid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ in a biological sample fromeach subject in a population of subjects determined not to have chronicfatigue syndrome (non-CFS subjects); b) calculating the amount of thepeptide relative to the total amount of protein in each sample of (a) todetermine a biomarker index for each subject in the population; c)establishing a threshold biomarker index from the biomarker indicesdetermined in (b); d) measuring the amount of a peptide comprising theamino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ in a biological samplefrom a test subject; and e) calculating the amount of the peptiderelative to the total amount of protein in the sample of (d) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than the threshold biomarkerindex of (c) identifies the subject as having chronic fatigue syndromeor having an increased likelihood of having or developing chronicfatigue syndrome.

In an additional aspect, the present invention provides a method ofidentifying a subject as having chronic fatigue syndrome or having anincreased likelihood of having or developing chronic fatigue syndrome,comprising: a) measuring the amount of a peptide comprising the aminoacid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ in a biological sample from eachsubject in a population of subjects determined not to have chronicfatigue syndrome (e.g., non-CFS subjects); b) calculating the amount ofthe peptide relative to the total amount of protein in each sample of(a) to determine a biomarker index for each subject in the population;c) establishing a threshold biomarker index from the biomarker indicesdetermined in (b); d) measuring the amount of a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ in a biological samplefrom a test subject; and e) calculating the amount of the peptiderelative to the total amount of protein in the sample of (d) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than the threshold biomarkerindex of (c) identifies the subject as having chronic fatigue syndromeor having an increased likelihood of having or developing chronicfatigue syndrome.

A further aspect of this invention is a method of identifying a subjectas having chronic fatigue syndrome or having an increased likelihood ofhaving or developing chronic fatigue syndrome, comprising: a) measuringthe amount of a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ in a biological sample from each subject ina population of subjects determined not to have chronic fatigue syndrome(e.g., non-CFS subjects); b) calculating the amount of the peptiderelative to the total amount of protein in each sample of (a) todetermine a biomarker index for each subject in the population; c)establishing a threshold biomarker index from the biomarker indicesdetermined in (b); d) measuring the amount of a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in a biological samplefrom a test subject; and e) calculating the amount of the peptiderelative to the total amount of protein in the sample of (d) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than the threshold biomarkerindex of (c) identifies the subject as having chronic fatigue syndromeor having an increased likelihood of having or developing chronicfatigue syndrome.

Further provided herein is a method of identifying a subject as havingchronic fatigue syndrome or having an increased likelihood of having ordeveloping chronic fatigue syndrome, comprising: a) measuring the amountof each of 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in a biological samplefrom each subject in a population of subjects determined not to havechronic fatigue syndrome (e.g., non-CFS subjects); b) calculating theamount of the peptides relative to the total amount of protein in eachsample of (a) to determine a biomarker index for each subject of thepopulation; c) establishing a threshold biomarker index from thebiomarker indices determined in (b); d) measuring the amount of eachof 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in a biological samplefrom a test subject; and e) calculating the amount of the peptidesrelative to the total amount of protein in the sample of (d) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than the threshold biomarkerindex of (c) identifies the subject as having chronic fatigue syndromeor having an increased likelihood of having or developing chronicfatigue syndrome.

Also provided herein is a method of identifying a subject as havingchronic fatigue syndrome or having an increased likelihood of having ordeveloping chronic fatigue syndrome, comprising: a) measuring the amountof two or more peptides selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ,2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising the amino acidsequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ, in a biological sample from eachsubject in a population of subjects determined not to have chronicfatigue syndrome; b) calculating the amount of the peptides relative tothe total amount of protein in each sample of (a) to determine abiomarker index for each subject of the population; c) establishing athreshold biomarker index from the biomarker indices determined in (b);d) measuring the amount of two or more peptides selected from the groupconsisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in a biological samplefrom a test subject (e.g., wherein the peptides of (d) are the same aspeptides as measured in (a)); and e) calculating the amount of thepeptides relative to the total amount of protein in the sample of (d) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than the threshold biomarkerindex of (c) identifies the subject as having chronic fatigue syndromeor having an increased likelihood of having or developing chronicfatigue syndrome.

An additional aspect of this invention includes a method of identifyinga subject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome, comprising:a) measuring the amount of human basic proline-rich protein 1 (PRB1) ina biological sample from each subject in a population of subjectsdetermined not to have chronic fatigue syndrome (e.g., non-CFSsubjects); b) calculating the amount of PRB1 relative to the totalamount of protein in each sample of (a) to determine a biomarker indexfor each subject in the population; c) establishing a thresholdbiomarker index from the biomarker indices determined in (b); d)measuring the amount of human basic proline-rich protein 1 (PRB1) in abiological sample from a test subject; and e) calculating the amount ofPRB1 relative to the total amount of protein in the sample of (d) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than the threshold biomarkerindex of (c) identifies the subject as having chronic fatigue syndromeor having an increased likelihood of having or developing chronicfatigue syndrome.

Other aspects of this invention include a method of identifying asubject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome, comprising:a) measuring the amount of human basic proline-rich protein 2 (PRB2) ina biological sample from each subject in a population of subjectsdetermined not to have chronic fatigue syndrome (e.g., non-CFSsubjects); b) calculating the amount of PRB2 relative to the totalamount of protein in each sample of (a) to determine a biomarker indexfor each subject in the population; c) establishing a thresholdbiomarker index from the biomarker indices determined in (b); d)measuring the amount of human basic proline-rich protein 2 (PRB2) in abiological sample from a test subject; and e) calculating the amount ofPRB2 relative to the total amount of protein in the sample of (d) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than the threshold biomarkerindex of (c) identifies the subject as having chronic fatigue syndromeor having an increased likelihood of having or developing chronicfatigue syndrome.

The present invention also provides a method of identifying a subject ashaving chronic fatigue syndrome or having an increased likelihood ofhaving or developing chronic fatigue syndrome, comprising: a) measuringthe amount of human basic proline-rich protein 4 (PRB4) in a biologicalsample from each subject in a population of subjects determined not tohave chronic fatigue syndrome (e.g., non-CFS subjects); b) calculatingthe amount of PRB4 relative to the total amount of protein in eachsample of (a) to determine a biomarker index for each subject in thepopulation; c) establishing a threshold biomarker index from thebiomarker indices determined in (b); d) measuring the amount of humanbasic proline-rich protein 4 (PRB4) in a biological sample from a testsubject; and e) calculating the amount of PRB4 relative to the totalamount of protein in the sample of (d) to determine a biomarker indexfor the test subject, wherein a biomarker index of the test subject thatis higher than the threshold biomarker index of (c) identifies thesubject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome.

Furthermore, the present invention provides a method of identifying asubject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome, comprising:a) measuring the amount of each of 1) human basic proline-rich protein 1(PRB1), 2) human basic proline-rich protein 2 (PRB2), and 3) human basicproline-rich protein 4 (PRB4) in a biological sample from each subjectin a population of subjects determined not to have chronic fatiguesyndrome (e.g., non-CFS subjects); b) calculating the amount of theproteins relative to the total amount of protein in each sample of (a)to determine a biomarker index for each subject in the population; c)establishing a threshold biomarker index from the biomarker indicesdetermined in (b); d) measuring the amount of 1) human basicproline-rich protein 1 (PRB1), 2) human basic proline-rich protein 2(PRB2), and 3) human basic proline-rich protein 4 (PRB4) in a biologicalsample from a test subject; and e) calculating the amount of theproteins relative to the total amount of protein in the sample of (d) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than the threshold biomarkerindex of (c) identifies the subject as having chronic fatigue syndromeor having an increased likelihood of having or developing chronicfatigue syndrome.

Additionally provided herein is a method of identifying a subject ashaving chronic fatigue syndrome or having an increased likelihood ofhaving or developing chronic fatigue syndrome, comprising: a) measuringthe amount of two or more proteins selected from the group consistingof 1) human basic proline-rich protein 1 (PRB1), 2) human basicproline-rich protein 2 (PRB2), and 3) human basic proline-rich protein 4(PRB4), in a biological sample from each subject in a population ofsubjects determined not to have chronic fatigue syndrome; b) calculatingthe amount of the proteins relative to the total amount of protein ineach sample of (a) to determine a biomarker index for each subject inthe population; c) establishing a threshold biomarker index from thebiomarker indices determined in (b); d) measuring the amount of two ormore proteins selected from the group consisting of: 1) human basicproline-rich protein 1 (PRB1), 2) human basic proline-rich protein 2(PRB2), and 3) human basic proline-rich protein 4 (PRB4), in abiological sample from a test subject, (e.g., wherein the proteins of(d) are the same proteins as measured in (a)); and e) calculating theamount of the proteins relative to the total amount of protein in thesample of (d) to determine a biomarker index for the test subject,wherein a biomarker index of the test subject that is higher than thethreshold biomarker index of (c) identifies the subject as havingchronic fatigue syndrome or having an increased likelihood of having ordeveloping chronic fatigue syndrome.

In further embodiments, the present invention provides a method ofidentifying a subject as having chronic fatigue syndrome or having anincreased likelihood of having or developing chronic fatigue syndrome,comprising: a) measuring the amount of a peptide comprising the aminoacid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ in a biological sample from atest subject; and b) calculating the amount of the peptide relative tothe total amount of protein in the sample of (a) to determine abiomarker index for the test subject, wherein a biomarker index of thetest subject that is higher than a threshold biomarker index identifiesthe subject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome.

In addition, the present invention provides a method of identifying asubject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome, comprising:a) measuring the amount of a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ in a biological sample from a test subject;and b) calculating the amount of the peptide relative to the totalamount of protein in the sample of (a) to determine a biomarker indexfor the test subject, wherein a biomarker index of the test subject thatis higher than a threshold biomarker index identifies the subject ashaving chronic fatigue syndrome or having an increased likelihood ofhaving or developing chronic fatigue syndrome.

Also provided herein is a method of identifying a subject as havingchronic fatigue syndrome or having an increased likelihood of having ordeveloping chronic fatigue syndrome, comprising: a) measuring the amountof a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ in a biological sample from a test subject;and b) calculating the amount of the peptide relative to the totalamount of protein in the sample of (a) to determine a biomarker indexfor the test subject, wherein a biomarker index of the test subject thatis higher than a threshold biomarker index identifies the subject ashaving chronic fatigue syndrome or having an increased likelihood ofhaving or developing chronic fatigue syndrome.

Furthermore, the present invention provides a method of identifying asubject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome, comprising:a) measuring the amount of 1) a peptide comprising the amino acidsequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the aminoacid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in a biological samplefrom a test subject; and b) calculating the amount of the peptidesrelative to the total amount of protein in the sample of (a) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than a threshold biomarkerindex identifies the subject as having chronic fatigue syndrome orhaving an increased likelihood of having or developing chronic fatiguesyndrome.

The present invention also provides a method of identifying a subject ashaving chronic fatigue syndrome or having an increased likelihood ofhaving or developing chronic fatigue syndrome, comprising: a) measuringthe amount of two or more peptides selected from the group consistingof: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ, in a biological samplefrom a test subject; and b) calculating the amount of the peptidesrelative to the total amount of protein in the sample of (a) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than a threshold biomarkerindex identifies the subject as having chronic fatigue syndrome orhaving an increased likelihood of having or developing chronic fatiguesyndrome.

Additionally, the present invention provides a method of identifying asubject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome, comprising:a) measuring the amount of human basic proline-rich protein 1 (PRB1) ina biological sample from a test subject; and b) calculating the amountof PRB1 relative to the total amount of protein in the sample of (a) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than a threshold biomarkerindex identifies the subject as having chronic fatigue syndrome orhaving an increased likelihood of having or developing chronic fatiguesyndrome.

In yet further embodiments, the present invention provides a method ofidentifying a subject as having chronic fatigue syndrome or having anincreased likelihood of having or developing chronic fatigue syndrome,comprising: a) measuring the amount of human basic proline-rich protein2 (PRB2) in a biological sample from a test subject; and b) calculatingthe amount of PRB2 relative to the total amount of protein in the sampleof (a) to determine a biomarker index for the test subject, wherein abiomarker index of the test subject that is higher than a thresholdbiomarker index identifies the subject as having chronic fatiguesyndrome or having an increased likelihood of having or developingchronic fatigue syndrome.

Further provided herein is a method of identifying a subject as havingchronic fatigue syndrome or having an increased likelihood of having ordeveloping chronic fatigue syndrome, comprising: a) measuring the amountof human basic proline-rich protein 4 (PRB4) in a biological sample froma test subject; and b) calculating the amount of PRB4 relative to thetotal amount of protein in the sample of (a) to determine a biomarkerindex for the test subject, wherein a biomarker index of the testsubject that is higher than a threshold biomarker index identifies thesubject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome.

The present invention also provides a method of identifying a subject ashaving chronic fatigue syndrome or having an increased likelihood ofhaving or developing chronic fatigue syndrome, comprising: a) measuringthe amount of 1) human basic proline-rich protein 1 (PRB1), 2) humanbasic proline-rich protein 2 (PRB2), and 3) human basic proline-richprotein 4 (PRB4) in a biological sample from a test subject; and b)calculating the amount of the proteins relative to the total amount ofprotein in the sample of (a) to determine a biomarker index for the testsubject, wherein a biomarker index of the test subject that is higherthan a threshold biomarker index identifies the subject as havingchronic fatigue syndrome or having an increased likelihood of having ordeveloping chronic fatigue syndrome.

Furthermore, the present invention provides a method of identifying asubject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome, comprising:a) measuring the amount of two or more proteins selected from the groupconsisting of: 1) human basic proline-rich protein 1 (PRB1), 2) humanbasic proline-rich protein 2 (PRB2), and 3) human basic proline-richprotein 4 (PRB4), in a biological sample from a test subject; and b)calculating the amount of the proteins relative to the total amount ofprotein in the sample of (a) to determine a biomarker index for the testsubject, wherein a biomarker index of the test subject that is higherthan a threshold biomarker index identifies the subject as havingchronic fatigue syndrome or having an increased likelihood of having ordeveloping chronic fatigue syndrome.

Additional aspects of this invention include a method of identifying adecrease over time in fatigue level of a subject, comprising: a)measuring, at a first time point, an amount of a peptide comprising theamino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ in a first biologicalsample from the subject; b) calculating the amount of the peptide of (a)relative to the total amount of protein in the first sample to determinea first biomarker index for the subject at the first time point; c)measuring, at a second or subsequent time point(s), an amount of thepeptide comprising an amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ,in a second or subsequent biological sample(s); and d) calculating theamount of the peptide of (c) relative to the total amount of the proteinin the second or subsequent sample(s) to determine a second orsubsequent biomarker index for the subject at the second or subsequenttime point(s), wherein a decrease in the second or subsequent biomarkerindex relative to the first biomarker index identifies a decrease overtime in the fatigue level of the subject.

The present invention further provides a method of identifying adecrease over time in fatigue level of a subject, comprising: a)measuring, at a first time point, an amount of a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ, in a first biologicalsample from the subject; b) calculating the amount of the peptide of (a)relative to the total amount of protein in the first sample to determinea first biomarker index for the subject at the first time point; c)measuring, at a second or subsequent time point(s), an amount of thepeptide comprising an amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ in asecond or subsequent biological sample(s); and d) calculating the amountof the peptide of (c) relative to the total amount of the protein in thesecond or subsequent sample(s) to determine a second or subsequentbiomarker index for the subject at the second or subsequent timepoint(s), wherein a decrease in the second or subsequent biomarker indexrelative to the first biomarker index identifies a decrease over time inthe fatigue level of the subject.

Also provided herein is a method of identifying a decrease over time infatigue level of a subject, comprising: a) measuring, at a first timepoint, an amount of a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ in a first biological sample from thesubject; b) calculating the amount of the peptide of (a) relative to thetotal amount of protein in the first sample to determine a firstbiomarker index for the subject at the first time point; c) measuring,at a second or subsequent time point(s), an amount of the peptidecomprising an amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in asecond or subsequent biological sample(s); and d) calculating the amountof the peptide of (c) relative to the total amount of the protein in thesecond or subsequent sample(s) to determine a second or subsequentbiomarker index for the subject at the second or subsequent timepoint(s), wherein a decrease in the second or subsequent biomarker indexrelative to the first biomarker index identifies a decrease over time inthe fatigue level of the subject.

Furthermore, the present invention provides a method of identifying adecrease over time in fatigue level of a subject, comprising: a)measuring, at a first time point, an amount of 1) a peptide comprisingthe amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQin a first biological sample from the subject; b) calculating the amountof the peptides of (a) relative to the total amount of protein in thefirst sample to determine a first biomarker index for the subject at thefirst time point; c) measuring, at a second or subsequent time point(s),an amount of 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in a second orsubsequent biological sample(s); and d) calculating the amount of thepeptides of (c) relative to the total amount of the protein in therespective sample(s) to determine a second or subsequent biomarker indexfor the subject at the second or subsequent time point(s), wherein adecrease in the second or subsequent biomarker index relative to thefirst biomarker index identifies a decrease over time in the fatiguelevel of the subject.

Furthermore, the present invention provides a method of identifying adecrease over time in fatigue level of a subject, comprising: a)measuring, at a first time point, an amount of two or more peptidesselected from the group consisting of: 1) a peptide comprising the aminoacid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptidecomprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in afirst biological sample from the subject; b) calculating the amount ofthe peptides of (a) relative to the total amount of protein in the firstsample to determine a first biomarker index for the subject at the firsttime point; c) measuring, at a second or subsequent time point(s), anamount of 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in a second orsubsequent biological sample(s) (e.g., wherein the peptides measured in(c) are the same peptides as measured in (a)); and d) calculating theamount of the peptides of (c) relative to the total amount of theprotein in the respective sample(s) to determine a second or subsequentbiomarker index for the subject at the second or subsequent timepoint(s), wherein a decrease in the second or subsequent biomarker indexrelative to the first biomarker index identifies a decrease over time inthe fatigue level of the subject.

In an additional aspect, the present invention provides a method ofidentifying a decrease over time in fatigue level of a subject,comprising: a) measuring, at a first time point, an amount of humanbasic proline-rich protein 1 (PRB1) in a first biological sample fromthe subject; b) calculating the amount of the protein of (a) relative tothe total amount of protein in the first sample to determine a firstbiomarker index for the subject at the first time point; c) measuring,at a second or subsequent time point(s), an amount of human basicproline-rich protein 1 (PRB1) in a second or subsequent biologicalsample(s); and d) calculating the amount of the protein of (c) relativeto the total amount of the protein in the second or subsequent sample(s)to determine a second or subsequent biomarker index for the subject atthe second or subsequent time point(s), wherein a decrease in the secondor subsequent biomarker index relative to the first biomarker indexidentifies a decrease over time in the fatigue level of the subject.

In a further aspect, the present invention provides a method ofidentifying a decrease over time in fatigue level of a subject,comprising: a) measuring, at a first time point, an amount of humanbasic proline-rich protein 2 (PRB2) in a first biological sample fromthe subject; b) calculating the amount of the protein of (a) relative tothe total amount of protein in the first sample to determine a firstbiomarker index for the subject at the first time point; c) measuring,at a second or subsequent time point(s), an amount of human basicproline-rich protein 2 (PRB2) in a second or subsequent biologicalsample(s); and d) calculating the amount of the protein of (c) relativeto the total amount of the protein in the second or subsequent sample(s)to determine a second or subsequent biomarker index for the subject atthe second or subsequent time point(s), wherein a decrease in the secondor subsequent biomarker index relative to the first biomarker indexidentifies a decrease over time in the fatigue level of the subject.

Further provided herein is a method of identifying a decrease over timein fatigue level of a subject, comprising: a) measuring, at a first timepoint, an amount of human basic proline-rich protein 4 (PRB4) in a firstbiological sample from the subject; b) calculating the amount of theprotein of (a) relative to the total amount of protein in the firstsample to determine a first biomarker index for the subject at the firsttime point; c) measuring, at a second or subsequent time point(s), anamount of human basic proline-rich protein 4 (PRB4) in a second orsubsequent biological sample(s); and d) calculating the amount of theprotein of (c) relative to the total amount of the protein in the secondor subsequent sample(s) to determine a second or subsequent biomarkerindex for the subject at the second or subsequent time point(s), whereina decrease in the second or subsequent biomarker index relative to thefirst biomarker index identifies a decrease over time in the fatiguelevel of the subject.

In an additional aspect of this invention, a method is provided, ofidentifying a decrease over time in fatigue level of a subject,comprising: a) measuring, at a first time point, an amount of 1) humanbasic proline-rich protein 1 (PRB1), 2) human basic proline-rich protein2 (PRB2), and 3) human basic proline-rich protein 4 (PRB4) in a firstbiological sample from the subject; b) calculating the amount of theproteins of (a) relative to the total amount of protein in the firstsample to determine a first biomarker index for the subject at the firsttime point; c) measuring, at a second or subsequent time point(s), anamount of 1) human basic proline-rich protein 1 (PRB1), 2) human basicproline-rich protein 2 (PRB2), and 3) human basic proline-rich protein 4(PRB4) in a second or subsequent biological sample(s); and d)calculating the amount of the proteins of (c) relative to the totalamount of the protein in the second or subsequent sample(s) to determinea second or subsequent biomarker index for the subject at the second orsubsequent time point(s), wherein a decrease in the second or subsequentbiomarker index relative to the first biomarker index identifies adecrease over time in the fatigue level of the subject.

In a further aspect of this invention, a method is provided, ofidentifying a decrease over time in fatigue level of a subject,comprising: a) measuring, at a first time point, an amount of two ormore proteins selected from the group consisting of: 1) human basicproline-rich protein 1 (PRB1), 2) human basic proline-rich protein 2(PRB2), and 3) human basic proline-rich protein 4 (PRB4) in a firstbiological sample from the subject; b) calculating the amount of theproteins of (a) relative to the total amount of protein in the firstsample to determine a first biomarker index for the subject at the firsttime point; c) measuring, at a second or subsequent time point(s), anamount of two or more proteins selected from the group consisting of: 1)human basic proline-rich protein 1 (PRB1), 2) human basic proline-richprotein 2 (PRB2), and 3) human basic proline-rich protein 4 (PRB4) in asecond or subsequent biological sample(s) (e.g., wherein the peptidesmeasured in (c) are the same peptides as measured in (a); and d)calculating the amount of the proteins of (c) relative to the totalamount of the protein in the second or subsequent sample(s) to determinea second or subsequent biomarker index for the subject at the second orsubsequent time point(s), wherein a decrease in the second or subsequentbiomarker index relative to the first biomarker index identifies adecrease over time in the fatigue level of the subject.

In yet further embodiments, the present invention provides a method ofguiding a human subject's treatment for chronic fatigue syndrome (CFS),comprising: a) measuring the concentration of a peptide selected fromthe group consisting of 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ,(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject; b) identifying the subject's level of chronic fatigueby calculating the ratio of the concentration of the peptide(s) measuredin (a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) having the subject initiate or resume a treatmentprogram for chronic fatigue syndrome; d) measuring the concentration ofa peptide selected from the group consisting of 1) a peptide comprisingthe amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) apeptide comprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ(SEQ ID NO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ, (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from the subject at one or more timepoints after (c), wherein the peptides of (d) are the same as thepeptides of (a); e) identifying the subject's level of chronic fatigueby calculating the ratio of the concentration of the peptide(s) measuredin (d) to the total amount of protein in the sample of (d) according tothe equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) guiding the subject's treatment for chronic fatiguesyndrome by modifying the intensity of subsequent treatments using thelatest of the subject's ratio(s) as calculated in (e), such that anincrease in chronic fatigue relative to the previous measurement ofchronic fatigue leads to a subsequent increase in the subject'streatment intensity.

Also provided herein is a method of evaluating the effect of a treatmentmaterial and/or activity on the chronic fatigue level of a human subject(e.g., a subject diagnosed as having, determined to have or suspected ofhaving chronic fatigue syndrome (CFS), comprising: a) measuring theconcentration of a peptide selected from the group consisting of 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), a 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from a subject at atime point prior to exposing the subject to a treatment material and/oractivity; b) identifying the subject's level of chronic fatigue bycalculating the ratio of the concentration of the peptide(s) measured in(a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) exposing the subject to the treatment material and/orperformance enhancing activity; d) measuring the concentration of apeptide selected from the group consisting of: 1) a peptide comprisingthe amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) apeptide comprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ(SEQ ID NO:2), a 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from the subject at one or more timepoints after (c), wherein the peptides of (d) are the same as thepeptides of (a); e) identifying the subject's level of chronic fatigueby calculating the ratio of the concentration of the peptide(s) measuredin (d) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) evaluating the effect of the treatment materialand/or activity on the chronic fatigue status of the subject bycomparing the ratios of (b) and (e), wherein an increase in the ratio of(e) relative to the ratio of (b) is indicative of increased chronicfatigue of the subject, a decrease in the ratio of (e) relative to theratio of (b) is indicative of decreased chronic fatigue of the subject,and a constant ratio of (e) relative to the ratio of (b) is indicativeof no change in chronic fatigue of the subject.

Additionally provided herein is a method of guiding a human subject'sphysical training activity, comprising: a) measuring the concentrationof a peptide selected from the group consisting of: 1) a peptidecomprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ IDNO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), a 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject whenthe subject is in a rested state, wherein the subject is an adultathlete or an amateur athlete; b) identifying the subject's level ofphysical fatigue by calculating the ratio of the concentration of thepeptide(s) measured in (a) to the total amount of protein in the sample,according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) having the subject initiate or resume a physicaltraining program comprising activities of different intensity; d)measuring the concentration of a peptide selected from the groupconsisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), a 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject at one or more time points after (c), wherein thepeptides of (d) are the same as the peptides of (a); e) identifying thesubject's level of physical fatigue by calculating the ratio of theconcentration of the peptide(s) measured in (d) to the total amount ofprotein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) guiding the subject's physical training program bymodifying the intensity of subsequent activities using the latest of thesubject's ratio(s), as calculated in (e), such that a decrease in theratio relative to the previous ratio leads to a subsequent increase inthe subject's training intensity, an increase in the ratio relative tothe previous ratio leads to a subsequent decrease in the subject'straining intensity, and a constant ratio relative to the previous ratioleads to a subsequent constant level in the subject's trainingintensity. In some embodiments, the physical training program can be amilitary training program.

The present invention also provides a method of evaluating the effect ofa performance enhancing material and/or activity on the physicalperformance capability of a human subject, comprising: a) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), a 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject at atime point prior to exposing the subject to a performance enhancingmaterial or performance enhancing activity, wherein the subject is anadult athlete or amateur athlete; b) identifying the subject's level ofphysical fatigue by calculating the ratio of the concentration of thepeptide(s) measured in (a) to the total amount of protein in the sample,according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) exposing the subject to the performance enhancingmaterial and/or performance enhancing activity; d) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), a 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject atone or more time points after (c), wherein the peptides of (d) are thesame as the peptides of (a); e) identifying the subject's level ofphysical fatigue by calculating the ratio of the concentration of thepeptide(s) measured in (d) to the total amount of protein in the sample,according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) evaluating the effect of the performance enhancingmaterial and/or activity on the physical performance capability of thesubject by comparing the ratios of (b) and (e), wherein an increase inthe ratio of (e) relative to the ratio of (b) is indicative of reducedphysical performance capability of the subject, a decrease in the ratioof (e) relative to the ratio of (b) is indicative of improved physicalperformance capability of the subject, and a constant ratio of (e)relative to the ratio of (b) is indicative of no change in physicalperformance capability of the subject.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “a” or “an” or “the” can mean one or more than one. Forexample, “a” cell can mean one cell or a plurality of cells.

Also as used herein, “and/or” refers to and encompasses any and/or allpossible combinations of one or more of the associated listed items, aswell as the lack of and and/or combinations when interpreted in thealternative (“or”).

Furthermore, the term “about” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even±0.1% of the specifiedamount.

The present invention is explained in greater detail below. Thisdescription is not intended to be a detailed catalog of all thedifferent ways in which the invention may be implemented, or all thefeatures that may be added to the instant invention. For example,features illustrated with respect to one embodiment may be incorporatedinto other embodiments, and features illustrated with respect to aparticular embodiment may be deleted from that embodiment. In addition,numerous variations and additions to the various embodiments suggestedherein will be apparent to those skilled in the art in light of theinstant disclosure, which do not depart from the instant invention.Hence, the following specification is intended to illustrate someparticular embodiments of the invention, and not to exhaustively specifyall permutations, combinations and variations thereof.

The present invention is based on the unexpected discovery of amodulation of biomarkers (e.g., modulation of peptide and/or proteinlevels) as measured in a biological sample from a subject that correlatewith the subject's status with regard to having chronic fatiguesyndrome, having an increased risk or likelihood of having or developingchronic fatigue syndrome and/or having an altered (e.g., increased ordecreased) level of fatigue over time.

Thus, in one embodiment, the present invention provides a method ofidentifying a subject as having chronic fatigue syndrome or having anincreased likelihood of having or developing chronic fatigue syndrome,comprising: a) measuring the amount of 1) a peptide comprising the aminoacid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and/or 3) a peptidecomprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ, in anycombination, in a biological sample from each subject in a population ofsubjects determined not to have chronic fatigue syndrome (e.g., non-CFSsubjects); b) calculating the amount of the peptide(s) relative to thetotal amount of protein in each sample of (a) to determine a biomarkerindex for each subject of the population; c) establishing a thresholdbiomarker index from the biomarker indices determined in (b); d)measuring the amount of 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and/or 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ, in any combination, ina biological sample from a test subject; and e) calculating the amountof the peptide(s) relative to the total amount of protein in the sampleof (d) to determine a biomarker index for the test subject, wherein abiomarker index of the test subject that is higher than the thresholdbiomarker index of (c) identifies the subject as having chronic fatiguesyndrome or having an increased likelihood of having or developingchronic fatigue syndrome.

In a further embodiment, the present invention provides a method ofidentifying a subject as having chronic fatigue syndrome or having anincreased likelihood of having or developing chronic fatigue syndrome,comprising: a) measuring the amount of 1) human basic proline-richprotein 1 (PRB1), 2) human basic proline-rich protein 2 (PRB2), and/or3) human basic proline-rich protein 4 (PRB4), in any combination, in abiological sample from each subject in a population of subjectsdetermined not to have chronic fatigue syndrome (e.g., non-CFSsubjects); b) calculating the amount of the protein(s) relative to thetotal amount of protein in each sample of (a) to determine a biomarkerindex for each subject in the population; c) establishing a thresholdbiomarker index from the biomarker indices determined in (b); d)measuring the amount of 1) human basic proline-rich protein 1 (PRB1), 2)human basic proline-rich protein 2 (PRB2), and/or 3) human basicproline-rich protein 4 (PRB4), in any combination, in a biologicalsample from a test subject; and e) calculating the amount of theprotein(s) relative to the total amount of protein in the sample of (d)to determine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than the threshold biomarkerindex of (c) identifies the subject as having chronic fatigue syndromeor having an increased likelihood of having or developing chronicfatigue syndrome.

In an additional embodiment, the present invention provides a method ofidentifying a decrease over time in fatigue level of a subject,comprising: a) measuring, at a first time point, an amount of 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ,2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ, and/or 3) a peptide comprising the amino acidsequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ, in any combination, in a firstbiological sample from the subject; b) calculating the amount of thepeptide(s) of (a) relative to the total amount of protein in the firstsample to determine a first biomarker index for the subject at the firsttime point; c) measuring, at a second or subsequent time point(s), anamount of 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and/or 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ, in any combination, ina second or subsequent biological sample(s); and d) calculating theamount of the peptide(s) of (c) relative to the total amount of theprotein in the second or subsequent sample(s) to determine a second orsubsequent biomarker index for the subject at the second or subsequenttime point(s), wherein a decrease in the second or subsequent biomarkerindex relative to the first biomarker index identifies a decrease overtime in the fatigue level of the subject.

Furthermore, the present invention provides a method of identifying asubject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome, comprising:a) measuring an amount of 1) a peptide comprising the amino acidsequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the aminoacid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ, in any combination, ina biological sample from a test subject; and b) calculating the amountof the peptide(s) relative to the total amount of protein in the sampleof (a) to determine a biomarker index for the test subject, wherein abiomarker index of the test subject that is higher than a thresholdbiomarker index identifies the subject as having chronic fatiguesyndrome or having an increased likelihood of having or developingchronic fatigue syndrome.

The present invention also provides a method of identifying a subject ashaving chronic fatigue syndrome or having an increased likelihood ofhaving or developing chronic fatigue syndrome, comprising: a) measuringan amount of 1) human basic proline-rich protein 1 (PRB1), 2) humanbasic proline-rich protein 2 (PRB2), and 3) human basic proline-richprotein 4 (PRB4), in any combination, in a biological sample from a testsubject; and b) calculating the amount of the protein(s) relative to thetotal amount of protein in the sample of (a) to determine a biomarkerindex for the test subject, wherein a biomarker index of the testsubject that is higher than a threshold biomarker index identifies thesubject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome.

In an additional embodiment of this invention, a method is provided, ofidentifying a decrease over time in fatigue level of a subject,comprising: a) measuring, at a first time point, an amount of 1) humanbasic proline-rich protein 1 (PRB1), 2) human basic proline-rich protein2 (PRB2), and/or 3) human basic proline-rich protein 4 (PRB4), in anycombination, in a first biological sample from the subject; b)calculating the amount of the protein(s) of (a) relative to the totalamount of protein in the first sample to determine a first biomarkerindex for the subject at the first time point; c) measuring, at a secondor subsequent time point(s), an amount of 1) human basic proline-richprotein 1 (PRB1), 2) human basic proline-rich protein 2 (PRB2), and/or3) human basic proline-rich protein 4 (PRB4) in a second or subsequentbiological sample(s); and d) calculating the amount of the protein(s) of(c) relative to the total amount of the protein in the second orsubsequent sample(s) to determine a second or subsequent biomarker indexfor the subject at the second or subsequent time point(s), wherein adecrease in the second or subsequent biomarker index relative to thefirst biomarker index identifies a decrease over time in the fatiguelevel of the subject.

In addition, the present invention provides a method of identifying anincrease over time in fatigue level of a subject, comprising: a)measuring, at a first time point, an amount of 1) a peptide comprisingthe amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and/or 3)a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ, in any combination, in a first biologicalsample from the subject; b) calculating the amount of the peptide(s) of(a) relative to the total amount of protein in the first sample todetermine a first biomarker index for the subject at the first timepoint; c) measuring, at a second or subsequent time point(s), an amountof 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and/or 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ, in any combination, ina second or subsequent biological sample(s); and d) calculating theamount of the peptide(s) of (c) relative to the total amount of theprotein in the second or subsequent sample(s) to determine a second orsubsequent biomarker index for the subject at the second or subsequenttime point(s), wherein an increase in the second or subsequent biomarkerindex relative to the first biomarker index identifies an increase overtime in the fatigue level of the subject.

In an additional embodiment of this invention, a method is provided, ofidentifying an increase over time in fatigue level of a subject,comprising: a) measuring, at a first time point, an amount of 1) humanbasic proline-rich protein 1 (PRB1), 2) human basic proline-rich protein2 (PRB2), and/or 3) human basic proline-rich protein 4 (PRB4), in anycombination, in a first biological sample from the subject; b)calculating the amount of the protein(s) of (a) relative to the totalamount of protein in the first sample to determine a first biomarkerindex for the subject at the first time point; c) measuring, at a secondor subsequent time point(s), an amount of 1) human basic proline-richprotein 1 (PRB1), 2) human basic proline-rich protein 2 (PRB2), and/or3) human basic proline-rich protein 4 (PRB4) in a second or subsequentbiological sample(s); and d) calculating the amount of the protein(s) of(c) relative to the total amount of the protein in the second orsubsequent sample(s) to determine a second or subsequent biomarker indexfor the subject at the second or subsequent time point(s), wherein anincrease in the second or subsequent biomarker index relative to thefirst biomarker index identifies an increase over time in the fatiguelevel of the subject.

Also provided herein is a method of identifying a subject as havingchronic fatigue syndrome or having an increased likelihood of having ordeveloping chronic fatigue syndrome, comprising: a) measuring the amountof two or more peptides selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ,2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising the amino acidsequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ, in a biological sample from eachsubject in a population of subjects determined not to have chronicfatigue syndrome; b) calculating the amount of the peptides relative tothe total amount of protein in each sample of (a) to determine abiomarker index for each subject of the population; c) establishing athreshold biomarker index from the biomarker indices determined in (b);d) measuring the amount of two or more peptides selected from the groupconsisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in a biological samplefrom a test subject (e.g., wherein the peptides of (d) are the same aspeptides as measured in (a)); and e) calculating the amount of thepeptides relative to the total amount of protein in the sample of (d) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than the threshold biomarkerindex of (c) identifies the subject as having chronic fatigue syndromeor having an increased likelihood of having or developing chronicfatigue syndrome.

Additionally provided herein is a method of identifying a subject ashaving chronic fatigue syndrome or having an increased likelihood ofhaving or developing chronic fatigue syndrome, comprising: a) measuringthe amount of two or more proteins selected from the group consistingof 1) human basic proline-rich protein 1 (PRB1), 2) human basicproline-rich protein 2 (PRB2), and 3) human basic proline-rich protein 4(PRB4), in a biological sample from each subject in a population ofsubjects determined not to have chronic fatigue syndrome; b) calculatingthe amount of the proteins relative to the total amount of protein ineach sample of (a) to determine a biomarker index for each subject inthe population; c) establishing a threshold biomarker index from thebiomarker indices determined in (b); d) measuring the amount of two ormore proteins selected from the group consisting of: 1) human basicproline-rich protein 1 (PRB1), 2) human basic proline-rich protein 2(PRB2), and 3) human basic proline-rich protein 4 (PRB4), in abiological sample from a test subject, (e.g., wherein the proteins of(d) are the same proteins as measured in (a)); and e) calculating theamount of the proteins relative to the total amount of protein in thesample of (d) to determine a biomarker index for the test subject,wherein a biomarker index of the test subject that is higher than thethreshold biomarker index of (c) identifies the subject as havingchronic fatigue syndrome or having an increased likelihood of having ordeveloping chronic fatigue syndrome.

The present invention also provides a method of identifying a subject ashaving chronic fatigue syndrome or having an increased likelihood ofhaving or developing chronic fatigue syndrome, comprising: a) measuringthe amount of two or more peptides selected from the group consistingof: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ, in a biological samplefrom a test subject; and b) calculating the amount of the peptidesrelative to the total amount of protein in the sample of (a) todetermine a biomarker index for the test subject, wherein a biomarkerindex of the test subject that is higher than a threshold biomarkerindex identifies the subject as having chronic fatigue syndrome orhaving an increased likelihood of having or developing chronic fatiguesyndrome.

Furthermore, the present invention provides a method of identifying asubject as having chronic fatigue syndrome or having an increasedlikelihood of having or developing chronic fatigue syndrome, comprising:a) measuring the amount of two or more proteins selected from the groupconsisting of: 1) human basic proline-rich protein 1 (PRB1), 2) humanbasic proline-rich protein 2 (PRB2), and 3) human basic proline-richprotein 4 (PRB4), in a biological sample from a test subject; and b)calculating the amount of the proteins relative to the total amount ofprotein in the sample of (a) to determine a biomarker index for the testsubject, wherein a biomarker index of the test subject that is higherthan a threshold biomarker index identifies the subject as havingchronic fatigue syndrome or having an increased likelihood of having ordeveloping chronic fatigue syndrome.

Furthermore, the present invention provides a method of identifying adecrease over time in fatigue level of a subject, comprising: a)measuring, at a first time point, an amount of two or more peptidesselected from the group consisting of: 1) a peptide comprising the aminoacid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptidecomprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in afirst biological sample from the subject; b) calculating the amount ofthe peptides of (a) relative to the total amount of protein in the firstsample to determine a first biomarker index for the subject at the firsttime point; c) measuring, at a second or subsequent time point(s), anamount of 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, 2) a peptide comprising the amino acidsequence GNPQGPSPQGGNKPQGPPPPPGKPQ, and 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ in a second orsubsequent biological sample(s) (e.g., wherein the peptides measured in(c) are the same peptides as measured in (a)); and d) calculating theamount of the peptides of (c) relative to the total amount of theprotein in the respective sample(s) to determine a second or subsequentbiomarker index for the subject at the second or subsequent timepoint(s), wherein a decrease in the second or subsequent biomarker indexrelative to the first biomarker index identifies a decrease over time inthe fatigue level of the subject.

In a further aspect of this invention, a method is provided, ofidentifying a decrease over time in fatigue level of a subject,comprising: a) measuring, at a first time point, an amount of two ormore proteins selected from the group consisting of: 1) human basicproline-rich protein 1 (PRB1), 2) human basic proline-rich protein 2(PRB2), and 3) human basic proline-rich protein 4 (PRB4) in a firstbiological sample from the subject; b) calculating the amount of theproteins of (a) relative to the total amount of protein in the firstsample to determine a first biomarker index for the subject at the firsttime point; c) measuring, at a second or subsequent time point(s), anamount of two or more proteins selected from the group consisting of: 1)human basic proline-rich protein 1 (PRB1), 2) human basic proline-richprotein 2 (PRB2), and 3) human basic proline-rich protein 4 (PRB4) in asecond or subsequent biological sample(s) (e.g., wherein the peptidesmeasured in (c) are the same peptides as measured in (a); and d)calculating the amount of the proteins of (c) relative to the totalamount of the protein in the second or subsequent sample(s) to determinea second or subsequent biomarker index for the subject at the second orsubsequent time point(s), wherein a decrease in the second or subsequentbiomarker index relative to the first biomarker index identifies adecrease over time in the fatigue level of the subject.

In yet further embodiments, the present invention provides a method ofguiding a human subject's treatment for chronic fatigue syndrome (CFS),comprising: a) measuring the concentration of a peptide selected fromthe group consisting of 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ,(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject; b) identifying the subject's level of chronic fatigueby calculating the ratio of the concentration of the peptide(s) measuredin (a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) having the subject initiate or resume a treatmentprogram for chronic fatigue syndrome; d) measuring the concentration ofa peptide selected from the group consisting of 1) a peptide comprisingthe amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) apeptide comprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ(SEQ ID NO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ, (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from the subject at one or more timepoints after (c), wherein the peptides of (d) are the same as thepeptides of (a); e) identifying the subject's level of chronic fatigueby calculating the ratio of the concentration of the peptide(s) measuredin (d) to the total amount of protein in the sample of (d) according tothe equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) guiding the subject's treatment for chronic fatiguesyndrome by modifying the intensity of subsequent treatments using thelatest of the subject's ratio(s) as calculated in (e), such that anincrease in chronic fatigue relative to the previous measurement ofchronic fatigue leads to a subsequent increase in the subject'streatment intensity.

Also provided herein is a method of evaluating the effect of a treatmentmaterial and/or activity on the chronic fatigue level of a human subject(e.g., a subject diagnosed as having, determined to have or suspected ofhaving chronic fatigue syndrome (CFS), comprising: a) measuring theconcentration of a peptide selected from the group consisting of 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), a 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from a subject at atime point prior to exposing the subject to a treatment material and/oractivity; b) identifying the subject's level of chronic fatigue bycalculating the ratio of the concentration of the peptide(s) measured in(a) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) exposing the subject to the treatment material and/orperformance enhancing activity; d) measuring the concentration of apeptide selected from the group consisting of: 1) a peptide comprisingthe amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) apeptide comprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ(SEQ ID NO:2), a 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from the subject at one or more timepoints after (c), wherein the peptides of (d) are the same as thepeptides of (a); e) identifying the subject's level of chronic fatigueby calculating the ratio of the concentration of the peptide(s) measuredin (d) to the total amount of protein in the sample, according to theequation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) evaluating the effect of the treatment materialand/or activity on the chronic fatigue status of the subject bycomparing the ratios of (b) and (e), wherein an increase in the ratio of(e) relative to the ratio of (b) is indicative of increased chronicfatigue of the subject, a decrease in the ratio of (e) relative to theratio of (b) is indicative of decreased chronic fatigue of the subject,and a constant ratio of (e) relative to the ratio of (b) is indicativeof no change in chronic fatigue of the subject.

Additionally provided herein is a method of guiding a human subject'sphysical training activity, comprising: a) measuring the concentrationof a peptide selected from the group consisting of: 1) a peptidecomprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ IDNO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), a 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject whenthe subject is in a rested state, wherein the subject is an adultathlete or an amateur athlete; b) identifying the subject's level ofphysical fatigue by calculating the ratio of the concentration of thepeptide(s) measured in (a) to the total amount of protein in the sample,according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) having the subject initiate or resume a physicaltraining program comprising activities of different intensity; d)measuring the concentration of a peptide selected from the groupconsisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), a 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject at one or more time points after (c), wherein thepeptides of (d) are the same as the peptides of (a); e) identifying thesubject's level of physical fatigue by calculating the ratio of theconcentration of the peptide(s) measured in (d) to the total amount ofprotein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) guiding the subject's physical training program bymodifying the intensity of subsequent activities using the latest of thesubject's ratio(s), as calculated in (e), such that a decrease in theratio relative to the previous ratio leads to a subsequent increase inthe subject's training intensity, an increase in the ratio relative tothe previous ratio leads to a subsequent decrease in the subject'straining intensity, and a constant ratio relative to the previous ratioleads to a subsequent constant level in the subject's trainingintensity. In some embodiments, the physical training program can be amilitary training program.

The present invention also provides a method of evaluating the effect ofa performance enhancing material and/or activity on the physicalperformance capability of a human subject, comprising: a) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), a 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject at atime point prior to exposing the subject to a performance enhancingmaterial or performance enhancing activity, wherein the subject is anadult athlete or amateur athlete; b) identifying the subject's level ofphysical fatigue by calculating the ratio of the concentration of thepeptide(s) measured in (a) to the total amount of protein in the sample,according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); c) exposing the subject to the performance enhancingmaterial and/or performance enhancing activity; d) measuring theconcentration of a peptide selected from the group consisting of: 1) apeptide comprising the amino acid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ(SEQ ID NO:1), 2) a peptide comprising the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), a 3) a peptide comprising theamino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4)any combination thereof, in a saliva sample taken from the subject atone or more time points after (c), wherein the peptides of (d) are thesame as the peptides of (a); e) identifying the subject's level ofphysical fatigue by calculating the ratio of the concentration of thepeptide(s) measured in (d) to the total amount of protein in the sample,according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ]+[GNPQGPSPQGGNKPQGPPPPPGKPQ]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ])/totalprotein (μg); and f) evaluating the effect of the performance enhancingmaterial and/or activity on the physical performance capability of thesubject by comparing the ratios of (b) and (e), wherein an increase inthe ratio of (e) relative to the ratio of (b) is indicative of reducedphysical performance capability of the subject, a decrease in the ratioof (e) relative to the ratio of (b) is indicative of improved physicalperformance capability of the subject, and a constant ratio of (e)relative to the ratio of (b) is indicative of no change in physicalperformance capability of the subject.

In methods wherein the level of more than one peptide or protein ismeasured for a subject, the biomarker index for the subject could bedetermined by any combination of the peptide or protein data. Forexample, the biomarker index might be a simple sum of the differentpeptide or protein levels, e.g., simple addition of the peptide orprotein levels and division of the sum by the total amount of protein inthe sample. More complex combinations of peptide or protein data mightalso be used. For example, the biomarker index could be calculated as aweighted sum of the various peptides or proteins, whereby the measuredpeptide or protein levels are multiplied by independent weightingfactors before the values are summed; it is possible that one of theweighting factors could be zero. Higher-order mathematical combinationsof peptide or protein levels might also be considered. For example, anequation for calculating the biomarker index might include a squared,cubed, or other higher-order term for one or more of the variouspeptides or proteins.

As used herein, “biomarker” can mean any chemical or biological entitythat is produced by cells and/or by commensal flora, or substances thatare produced by cells or commensal flora that might be then chemicallymodified by extracellular enzymes, free radicals produced by cells ofthe body and/or other naturally occurring processes and that is found,for example, in the saliva, urine, blood, vaginal secretion, tears,feces, sputum, hair, nails, skin, wound fluid, nasal swab, lymph,perspiration, oral mucosa, vaginal mucosa, or the anus, or in serum orplasma obtained from blood. Thus, in the methods of this invention, thesample can be any biological fluid or tissue that can be used in anassay of this invention, including but not limited to, serum, plasma,blood, saliva, semen, lymph, cerebrospinal fluid, prostatic fluid,urine, sputum, oral mucosa, nasal mucosa, duodenal fluid, gastric fluid,skin, endothelium, biopsy material from a salivary gland, biopsymaterial of a parotid gland, biopsy material of other glands of themouth, secretions of the salivary gland, secretions of the parotidgland, secretions of other glands of the mouth, joint fluid, body cavityfluid, tear fluid, anal secretions; vaginal secretions, perspiration,whole cells, cell extracts, tissue, biopsy material, aspirates,exudates, slide preparations, fixed cells, tissue sections, etc.

In various embodiments of this invention, the biological sample can beprepared according to methods well known in the art and as described inthe Examples section herein, to be a small molecular weight (SMW)sample. In particular embodiments of this invention, the biologicalsample is saliva.

In some embodiments, a biomarker of this invention can be, but is notlimited to, a peptide or polypeptide comprising, consisting essentiallyof and/or consisting of the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ, the amino acid sequenceGNPQGPSPQGGNKPQGPPPPPGKPQ, and/or the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ, singly or in any combination. In someembodiments, the biomarker of this invention can be, but is not limitedto human basic proline-rich protein 1 (PRB1), human basic proline-richprotein 2 (PRB2), and/or 3) human basic proline-rich protein 4 (PRB4),the amino acid sequence and nucleotide sequence of each of which isknown in the art, as exemplified in Table 2.

In further embodiments, a biomarker of this invention can be a peptidecomprising, consisting essentially of or consisting of any fragment ofhuman basic proline-rich protein 1 (PRB1), human basic proline-richprotein 2 (PRB2), and/or 3) human basic proline-rich protein 4 (PRB4).For example, the biomarker of this invention can be a peptidecomprising, consisting essentially of or consisting of one or more 5mers, 6 mers, 7 mers, 8 mers, 9 mers or 10 mers of the amino acidsequence of any of human basic proline-rich protein 1 (PRB1), humanbasic proline-rich protein 2 (PRB2), and/or 3) human basic proline-richprotein 4 (PRB4).

As a nonlimiting example, a biomarker of this invention can be a peptidecomprising, consisting essentially of or consisting of at least aboutfive amino acids (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) to at leastabout 15, 20, 25, 30, 35, 40, 45, 50, 50, 70, 80, 90, 100, 125, 150, 175or 200 amino acids (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112,113, 114, 115, 116, 117, 118, 119, 1120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140,141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154,155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, etc.), wherein the peptide comprises one or more(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or20) of the 5 mer peptides listed in Table 1. As a nonlimiting example, abiomarker peptide of this invention can be 20 amino acids in length andcan comprise two of the 5 mer peptides of Table 1, which can becontiguous as provided in the protein sequence from which the 5 merpeptides were derived. The remaining 10 amino acids can be on eitherside of each 5 mer, between each 5 mer or both. Furthermore, theremaining amino acids can be amino acids that are contiguous with the 5mer peptides and/or noncontiguous with the 5 mer peptides (e.g., aminoacids that are not present in the order provided in the protein sequencefrom which the 5 mer peptides were derived). The 5 mers of the PRBproteins provided in Table 1 are exemplary, as the present inventionalso encompasses 6 mers, 7 mers, 8 mers, 9 mers, etc., of these PRBproteins as noted above, the amino acid sequence of any of which wouldbe readily determined by one of skill in the art.

In further embodiments, a biomarker of this invention can comprise,consist essentially of or consist of any peptide or protein listed inTable 1, singly or in any combination. The biomarker of this inventioncan also comprise, consist essentially of or consist of any fragment ofany peptide or protein listed in Table 1.

Thus, in certain embodiments, the present invention is directed to abiomarker, which is a peptide or polypeptide (i.e., protein) asdescribed herein and the present invention can employ or involve one ormore of the peptides and proteins set forth herein in any method and/orkit of this invention, singly and/or in any combination. In certainother embodiments, the present invention provides a nucleic acidencoding a biomarker of this invention and the methods of this inventioncan employ or involve one or more of the nucleic acids of this inventionin any method/and or kit of this invention.

A biomarker of this invention can be detected and/or quantified in asample by a variety of methods well known in the art for detectingand/or quantifying substances in biological samples. For example, fordetecting and/or quantifying a biomarker that is a peptide orpolypeptide, standard methods for detecting and/or quantifying peptidesand/or polypeptides in sample can be employed. Nonlimiting examples ofsuch methods include direct protein measurement, absorbance at 280 nm,absorbance at 205 nm, extinction coefficient assay, Lowry assay, biuretassay, Bradford assay, bicinchoninic acid assay (BCA), amido blackassay, colloidal gold assay, immunoassay or other specific binding assayemploying an antibody or ligand that specifically binds a peptide orpolypeptide, protein separation assays such as electrophoresis, gaschromatography (GC), high performance liquid chromatography (HPLC), massspectrometry (MS), etc., as are well known in the art.

Other methods of detection can include bioassays using mammalian orbacterial cells wherein an output is proportional to the concentrationof peptide in the sample solution, and solid phase methods whereinbinding to a surface coated with a peptide recognizing molecule triggersan output electrical signal or change in optical property.

In some embodiments of this invention, peptides and/or proteins in asample of this invention can be measured by BCA.

For detection and/or quantification of a nucleic acid encoding abiomarker of this invention, standard methods for detection and/orquantification of nucleic acids in a sample can be employed.Non-limiting examples include hybridization assays, amplificationassays, sequencing protocols, etc., as are well known in the art.

The term “biomarker index” as used herein means the ratio of the amountof biomarker peptide or protein to the total amount of protein in thebiological sample. For example, the biomarker index can be a valuedetermined by calculating the relative amount of peptide in nmoles perμg of total protein (e.g., as determined by BCA) in the biologicalsample. The term “threshold biomarker index” as used herein means thebiomarker index calculated from the biomarker indices from a populationof subjects that defines the threshold value for identifying a subjectas having chronic fatigue syndrome or having an increased likelihood orrisk of having or developing chronic fatigue syndrome. The thresholdbiomarker index is calculated from the biomarker index of each subjectin a population of subjects determined not to have chronic fatiguesyndrome (e.g., non-CFS subjects). In some embodiments, a predeterminedor previously established threshold biomarker index can be used in themethods described herein to identify a subject as having chronic fatiguesyndrome or having an increased likelihood of having or developingchronic fatigue syndrome. Such a predetermined or previously establishedthreshold biomarker index can be determined according to the teachingsset forth herein (see, e.g., Example 3) as well as art-known teachings.

A subject of this invention is any animal in which identification ofchronic fatigue syndrome, identification of increased likelihood or riskof having or developing chronic fatigue syndrome and/or identificationof changes in fatigue level over time is needed or desired. In someembodiments, the subject is mammal and in particular embodiments thesubject is a human. In other embodiments, the subject can be a horse, adog or any other mammal about which the information obtained from themethods of this invention is needed or desired. In some embodiments, thesubject can be diagnosed with chronic fatigue syndrome and/or havesymptoms of chronic fatigue syndrome or other fatigue-related disorderand in some embodiments the subject can have no diagnosis or symptom(s)of chronic fatigue syndrome or any other fatigue-related disorder. Asubject of this invention that has an “increased likelihood” or“increased risk” of having or developing chronic fatigue syndrome can bea subject having symptoms and/or signs of chronic fatigue syndrome orother fatigue associated disorder or such a subject can be a subject whois not having symptoms and/or signs of chronic fatigue syndrome or otherfatigue-associated disorder. A subject of this invention can have anincreased risk or increased likelihood of having or developing chronicfatigue syndrome due to environmental and/or genetic factors as would beknown to one of skill in the art. By “increased likelihood” or“increased risk” of having or developing chronic fatigue syndrome it ismeant that the increase is relative to a control (e.g., a subject whosebiomarker index is at or below the threshold biomarker index).

In the methods of this invention employing measurements over time, thetime intervals can be minutes, hours, days, weeks, months and/or yearsin any order and in any combination.

In the methods of this invention that recite a first time point and asecond or subsequent time points or later time points, in someembodiments, the first time point can be prior to performance of anactivity (e.g., physical, athletic, mental, training, normal activity ofdaily living, etc.) and the second or subsequent time points can beduring and/or after performance of the activity. In some embodiments,the first time point can be during and/or after the performance ofactivity and the second or subsequent points can be at a later timefollowing completion of the activity.

Physical activity and/or athletic activity as described herein can bebut is not limited to ultra-endurance exercise, a military operation,military training, running, walking, bicycling, weight lifting,swimming, a standardized physical test course including, for example,those used by the military, a triathlon, a biathlon, shooting of arifle, shooting of a handgun, the aiming of computerized targetequipment, staying awake, hiking, hiking while carrying a large burdenon the back, physical activities of daily living and any combinationthereof.

As used herein, the term “ultra-endurance exercise” means a singlecontinuous session of physical activity and/or athletic activity duringwhich the subject performs said activity for a minimum of four hourswith an average exertion equal to or greater than 70% of ventalitorythreshold, as described in Harger-Domitrovich et al., 2007, Medicine &Science in Sports & Exercise. For example, ten hours of continuousrepetition of a one-hour exercise regimen consisting of 9 min. ofupper-body ergometry, 19 min. of cycling, and 20 min. of treadmillwalking with 1-min transition between modes, followed by a 10-min. restand feeding period.

Military training is defined as the process of preparing militaryindividuals and units to perform their assigned functions and missions,particularly to prepare for combat and wartime functions. “Coveringevery aspect of military activity, training is the principal occupationof military forces when not actually engaged in combat.” (Brassey'sEncyclopedia of Land Forces and Warfare, By Franklin D. Margiotta).Training may include for example physical tasks such as swimming, hikingwhen equipped with full military gear, running, moving stealthily,climbing, performing the aforementioned tasks under extremeenvironmental conditions such as high and low temperatures, highaltitude or under conditions where flora and fauna pose significanthazard.

Military operations are defined for the purposes of this instantinvention as the activities engaged in by soldiers, sailors and airmenduring performance of duties during periods of war and peace. Militaryoperations may include tasks related to engagement of enemy combatants.These tasks may include, but are not limited to the following examples,pursuing the enemy on foot, flying unmanned drone aircraft, operatingelectronic equipment, manning guns in flight on an airplane, performinglaw enforcement duties and providing intelligence.

Normal activities of daily living, as defined by the National CancerInstitute, include eating, dressing, getting into or out of a bed orchair, taking a bath or shower, and using the toilet. Instrumentalactivities of daily living are activities related to independent livingand include preparing meals, managing money, shopping, doing housework,and using a telephone.

The terms “fatigue” and “fatigued state” as used herein mean wearinessor exhaustion from labor, exertion, exercise, or stress, including lossof physical strength and bodily and mental capabilities.

The term “chronic fatigue” as used herein describes fatigue lastingabout six or more consecutive months, which is not due or identified tobe due to ongoing exertion or other medical conditions associated withfatigue.

“Chronic fatigue syndrome” or “CFS” as used herein describes thecondition or status of a subject (e.g., a patient) who meets at leastone of the criteria set forth for example, in 1) the CDC definition(1994) and also called the Fukuda definition, 2) The Oxford criteria(1991), which includes CFS of unknown etiology and a subtype calledpost-infectious fatigue syndrome (PIFS), 3) The 2003 Canadian Clinicalworking definition, which states that “[a] patient with ME/CFS will meetthe criteria for fatigue, post-exertional malaise and/or fatigue, sleepdysfunction, and pain; have two or more neurological/cognitivemanifestations and one or more symptoms from two of the categories ofautonomic, neuroendocrine, and immune manifestations; and the illnesswill persist for at least 6 months,” 4) CFS/ME guideline for theNational Health Service in England and Wales, produced in 2007 by theNational Institute for Health and Clinical Excellence (NICE), and 5)other criteria including gene expression markers, genetic profilesand/or biomarkers as are known in the art. See, e.g., “Chronic FatigueSyndrome/Myalgic Encephalomyelitis, A Primer for Clinical Practitioners”by the International Association for Chronic Fatigue Syndrome/MyalgicEncephalomyelitis (IACFS/ME), 2012 Edition, the entire contents of whichare incorporated by reference herein. See also the following websites:www.iacfsme.org and www.cdc.gov/cfs/general/index.html, the entirecontents of each of which are incorporated by reference herein.

A “treatment program for chronic fatigue syndrome” describes aphysician-directed program including one or more of the treatments forChronic Fatigue Syndrome recommended by the Centers for Disease Controland Prevention, International Association for Chronic FatigueSyndrome/Myalgic Encephalomyelitis, and/or other recognizedorganizations with expertise in Chronic Fatigue Syndrome

As used herein, “increasing the intensity of a treatment program” meanschanging a subject's treatment program such that the supervisingphysician believes that the level or intensity of future treatment isgreater than the previous level or intensity of treatment.

Nonlimiting examples of treatments for chronic fatigue syndrome (CFS)include 1) cognitive behavioral therapy, 2) graded exercise therapy as aform of physical therapy, 3) pacing or energy management therapy, 4)pharmacotherapy including, e.g., Amitriptyline, Doxepin, Nortriptyline,Cyclobenzaprine, Trazodone, Gabapentin, Pregabalin, Promethazine,Diphenhydramine, Clonazepam, Orphenadrine, Ropinirole, pramipexole,Melatonin, Zolpidem, Zopiclone, Mirtazapine, Acetaminophen, Aspirin,Diclofenac, Gabapentin, Duloxetine, Codeine phosphate, oxycodone,hydrocodone, morphine, Tramadol, Modafinil, Armnodafanil,Methylphenindate, Dexamphetamine, Caffeine, Amphetamine saltsIsoprinosine, anti-viral medications, rintatolimod, rituximab,antibiotics, anti-parasitics, dietary supplementation, vitamin D,vitamin B-12, B-complex vitamins, essential fatty acids, zinc and herbalremedies, light and radiation therapy, and any combination of theseagents and therapies.

Also as used herein, the term “rested” or “rested state” or“non-fatigued state” means having sufficient rest from bodily and/ormental exertion, either before physical exercise and/or after recoveryfrom fatigue.

“Perceived level of fatigue” as used herein means an individual'spersonal estimate or assessment of their fatigue and their ability tocarry out tasks requiring a certain level of physical and/or cognitiveperformance

In addition, as used herein, “physical performance capability” means thecapacity to accomplish a task which requires expenditure of a particularamount of energy. These tasks include, but are not limited to liftingobjects, carrying objects, maintaining a certain pace of walking,running and/or cycling and maintaining a particular heart rate for aspecified period. Capability represents a subject's potential for energyexpenditure over a particular period of time.

The term “fitness” as used herein means good health or physicalcondition, especially as the result of exercise and proper nutrition.

In some embodiments of this invention, a subject of this invention isany animal in which identification of chronic fatigue syndrome,identification of increased likelihood or risk of having or developingchronic fatigue syndrome and/or identification of changes in fatiguelevel over time is needed or desired. In some embodiments, the subjectis mammal and in particular embodiments the subject is a human. In otherembodiments, the subject can be a horse, a dog or any other mammal aboutwhich the information obtained from the methods of this invention isneeded or desired.

In some embodiments, the subject can be diagnosed with chronic fatiguesyndrome and/or have symptoms of chronic fatigue syndrome or otherfatigue-related disorder and in some embodiments the subject can have nodiagnosis or symptom(s) of chronic fatigue syndrome or any otherfatigue-related disorder. A subject of this invention that has an“increased likelihood” or “increased risk” of having or developingchronic fatigue syndrome can be a subject having symptoms and/or signsof chronic fatigue syndrome or other fatigue associated disorder or sucha subject can be a subject who is not having symptoms and/or signs ofchronic fatigue syndrome or other fatigue-associated disorder. A subjectof this invention can have an increased risk or increased likelihood ofhaving or developing chronic fatigue syndrome due to environmentaland/or genetic factors as would be known to one of skill in the art. By“increased likelihood” or “increased risk” of having or developingchronic fatigue syndrome it is meant that the increase is relative to acontrol (e.g., a subject whose biomarker index is at or below thethreshold biomarker index).

In some embodiments, a population of study subjects of this inventionincludes healthy male and/or female volunteers less than the age of 42that are in good physical condition and not suffering from knowndiseases and/or healthy young (less than 25 years old) military membersbeing screened for selection to Special Forces in the United StatesMilitary.

In some embodiments of this invention, a subject of this invention canbe military personnel, a shift worker, a laborer, a truck driver,airline personnel, assembly line worker, a patient at a sleep clinic, anamateur athlete, a professional athlete, a worker in the oil and/or gasindustry, a train driver, an astronaut, a space traveler, a coal miner,an air traffic controller and any combination thereof. A subject of thisinvention can also be any subject engaged in training and/or engaged inathletics, other employment or other activities outside of definedmilitary duties. A subject of this invention may also be engaged in anywork occupation as listed in the Standard Occupational Classification(SOC) System Manual, Version 2010, published by the US Department ofCommerce and/or Brassey's Encyclopedia of Land Forces and Warfare, ByFranklin D. Margiotta (1996) and/or The International Classification ofSleep Disorders Revised (2001) (ISBN-10: 157488087X) and/or “SleepDisorders and Sleep Deprivation: An Unmet Public Health Problem” HarveyR. Colten and Bruce M. Altevogt, Editors, Committee on Sleep Medicineand Research, ISBN: 0-309-65727-X, 424 pages, (2006), the entirecontents of each of which are incorporated by reference herein.

As used herein, a “sleep schedule” refers to periods of time allocatedto sleep and taken at a specific time over some defined and recurringperiod. This can mean, as a non-limiting example, allocating a sleepperiod of 8 hours starting at 10 PM where the recurring period is 24hours long where each recurring period starts at 6 AM. In anotherexample one sleep period is 2 hours and starts at 4 PM and a secondperiod starts at lAM and is 4 hours long where the recurring period is24 hours long where each recurring period starts at 6 AM.

Guiding a subject's sleep schedule can be done by modifying the durationof the sleep periods, the number of sleep periods per day, and/or thetime at which sleep periods are started. The recurring period may bedetermined, for example, by the subject's work schedule and/or anyconvenient recurring time pattern for example per day, week, month, etc.

Also as used herein, a “sleep enhancing material” refers to foods thatare ingested, drugs that are administered by the oral route, intravenousroute, transdermal route, intranasal route, via inhalation, byintra-ocular route, by vaginal route, and/or by rectal delivery route orother means, or complex mixtures that are delivered through acombination of routes in the form of a bath or immersion. Nonlimitingexamples of a sleep enhancing material include an herbal tea, warm milk,turkey, a large meal, an alcoholic beverage, a dietary supplement, a mudbath, a salt bath, an FDA-approved drug agent with a specific indicationfor treatment of a sleep disorder including, e.g., insomnia, narcolepsy,sleep apnea, depression, anxiety, and sleep disorder associated withshift work.

Furthermore, a “sleep enhancing activity” refers to actions taken by thesubject to promote sleepiness and thereby sleep, and/or actions taken byothers directed towards the subject to promote sleepiness and therebysleep, and/or actions taken by an automated system directed towards thesubject to promote sleepiness and thereby sleep. Nonlimiting examples ofa sleep enhancing activity include reading while lying down, readingpoetry, watching television, listening to music, exposure toelectromagnetic fields, having sex, soaking in warm water, massage,counting sheep and/or other redundant mental activity.

As used herein, “treatment of a sleep disorder” includes actions takenby a medical doctor or other medical practitioner on the subject toreduce symptoms associated with a sleep disorder or actions taken by anon-medical therapist or counselor on the subject to treat a sleepdisorder or action taken by the spouse or friend or acquaintance of thesubject to treat a sleep disorder or action taken by the subject totreat a sleep disorder. Nonlimiting examples of a treatment of a sleepdisorder include taking an FDA approved agent and/or using a device fortreatment of sleep disorder as prescribed by the subject's physician,psychological therapy delivered by a therapist to the subject to reduceanxiety and thereby mitigate sleep disorder, cognitive engagement by aspouse to improve marital relationship thereby reducing sleep disorder,a commitment and/or action on the part of the subject to work lessthereby reducing sleep disorder. Enhancement of a treatment of a sleepdisorder means increased intensity and/or frequency and/or dose and/oradding a new or additional treatment to an existing treatment regimenReduction of a treatment of a sleep disorder means reduced intensity orfrequency and/or dose and/or removing a treatment from an existingtreatment regimen.

“Treatment of fatigue” as used herein means medical interventions and/orother physical and/or mental actions and/or changes in behavior toreduce fatigue. Nonlimiting examples of a treatment of fatigue includethe use of medical interventions that may include drugs, surgery, and/oruse of a approved medical device and/or approved medical therapyintended to reduce fatigue, the use of cognitive therapy and/orcounseling to promote or elicit behaviors that reduce fatigue byreducing exposure to fatiguing activities and/or promoting activitiesthat reduce fatigue. Specific non-limiting examples include taking aprescribed drug to promote restful sleep, thereby reducing fatigue,and/or engaging in fatigue mitigation counseling that leads, forexample, to scheduling of more time for sleep.

As used herein, the term “work load” refers to cognitive and/or physicaltasks that are required and/or desired for living. This includes tasksthat are performed at home and outside the home. This includes, forexample tasks related to activities of daily living and/or tasksaccomplished or performed at a subject's workplace.

As also used herein, “fitness for duty” means having the ability toperform cognitive and/or physical tasks associated with daily livingand/or work and having the ability to perform these tasks withinreasonable periods of time and at a certain level of quality.

A “threshold ratio” describes a level of the ratio that is associatedwith a high likelihood of being able to perform a specific physicaland/or cognitive task and/or to be fit for duty or to be in anon-fatigued state.

To perform a physical activity and/or athletic activity at a sufficientlevel means the ability to perform a task at a level that is requiredfor professional advancement, required to pass a test, and/or requiredto complete a study. This also may include but is not limited to meetingindividual personal physical and performance goals, satisfying jobeligibility requirements, and/or meeting criteria required to continueworking at a task, for example determining whether a person is toofatigued to drive a truck.

In some embodiments, to be “sufficiently rested to carry out a workload” includes having the ability to perform a task at a level that isrequired for professional advancement, required to pass a test and/ormeet a predetermined threshold of performance and/or required tocomplete a study.

In some embodiments, a population of study subjects of this inventionincludes healthy male and/or female volunteers less than the age of 42that are in good physical condition and not suffering from knowndiseases (e.g., determined not to have chronic fatigue syndrome) and/orhealthy young (less than 25 years old) military members being screenedfor selection to Special Forces in the United States Military.

To perform a physical activity and/or athletic activity at a sufficientlevel means the ability to perform a task at a level that is requiredfor professional advancement, required to pass a test, and/or requiredto complete a study. This also may include but is not limited to meetingindividual personal physical and performance goals, satisfying jobeligibility requirements, and/or meeting criteria required to continueworking at a task, for example determining whether a person is toofatigued to drive a truck.

In some embodiments, to be “sufficiently rested to carry out a workload” includes having the ability to perform a task at a level that isrequired for professional advancement, required to pass a test and/ormeet a predetermined threshold of performance and/or required tocomplete a study.

The term “chronic fatigue syndrome” as used herein describes anart-known syndrome, the signs and symptoms of which are described in theliterature (see, e.g., Reeves et al. “Prevalence of chronic fatiguesyndrome in metropolitan, urban, and rural Georgia” Population HealthMetrics 5:5 (2007), the entire contents of which are incorporated byreference herein). Common symptoms and signs of chronic fatigue syndromeinclude fatigue, loss of memory or concentration, sore throat, enlargedlymph nodes in the neck and/or armpits, unexplained muscle pain, painthat moves from one joint to another without swelling or redness,headache of a new type, pattern or severity, unrefreshing sleep, andextreme exhaustion lasting more than 24 hours after physical or mentalexercise. To meet the diagnostic criteria of chronic fatigue syndrome, asubject typically must have unexplained, persistent fatigue for sixmonths or more, along with at least four of the following signs andsymptoms: loss of memory or concentration, sore throat, enlarged lymphnodes in the neck or armpits, unexplained muscle pain, pain that movesfrom one joint to another without swelling or redness, headache of a newtype, pattern or severity, unrefreshing sleep, and extreme exhaustionlasting more than 24 hours after physical or mental exercise.

The biomarkers and biomarker indices of this invention are correlatedwith chronic fatigue syndrome, fatigue, a fatigued state, an increase ordecrease in fatigue, an increase or decrease in physical performance, asubject's perceived level of fatigue, recovery from a fatigued state,and/or an increased or decreased likelihood of performing an activity ata sufficient level as described herein according to methods well knownin the art and as disclosed in the Examples provided herein. In general,identifying such correlation involves conducting analyses that establisha statistically significant association and/or a statisticallysignificant correlation between the presence of a biomarker or biomarkerindex or a combination of biomarkers or biomarker indices and a changein the subject (e.g., from rested to fatigued state, during and/or afterperformance of physical activity or other defined or standardizedactivity) as detected according to standard methods. An analysis thatidentifies a statistical association (e.g., a significant association)between the biomarker or biomarker index or between the combination ofbiomarkers or biomarker indices and the change in the subjectestablishes a correlation between the increase or decrease of thebiomarker or biomarker index or combination of biomarkers or biomarkerindices in a subject and the change being analyzed.

It would be well understood by one of skill in the art that the methodsof the present invention can be carried out on multiple subjects and thedata compiled to produce mean and median values that indicate fatigue, afatigued state, an increase or decrease in fatigue, an increase ordecrease in physical performance, a subject's perceived level offatigue, recovery from a fatigued state, and/or an increased ordecreased likelihood of performing a physical activity at a sufficientlevel according to this invention. It would also be understood that thestatistical limits described by the data obtained from groups ofsubjects can be applied to individual subjects' response. Thus, it wouldbe understood that in some embodiments of this invention, the methods ofthis invention can be carried out using a computer, wherein, forexample, the data from multiple subjects are stored in a computerdatabase and analyzed according to art-known methods of statistical andmathematical analysis to identify means, medians, trends, statisticallysignificant changes, variances, etc.

Thus, in some embodiments, the methods of this invention can be carriedout using a computer. Thus the present invention provides acomputer-assisted method of identifying fatigue, a fatigued state, anincrease or decrease in fatigue, an increase or decrease in physicalperformance, a subject's perceived level of fatigue, recovery from afatigued state, and/or an increased or decreased likelihood ofperforming a physical activity at a sufficient level. The methodinvolves the steps of (a) storing a database of biological data for aplurality of subjects, the biological data that is being storedincluding for each of said plurality of subjects: (i) a description ofthe status of the subject and/or physical/athletic activity performed bythe subject, (ii) a description of any performance enhancing materialand/or activity administered to, contacted with and/or implemented bythe subject; (iii) a description of measurements according to art-knownmethods detecting a change in the status or performance in the subject;and (iv) a description of measurements of biomarkers or biomarkerindices in the subject; and then (b) querying the database to determinethe relationship between a change in the measurement of biomarkers orbiomarker indices in the subject and change in performance or status ofthe subject. Such querying can be carried out prospectively orretrospectively on the database by any suitable means, but is generallydone by statistical analysis in accordance with known techniques, asdescribed herein.

Compositions of the Invention

Further aspects of the present invention include an isolated peptidecomprising, consisting essentially of, or consisting of the amino acidsequence of PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), an isolatedpeptide comprising, consisting essentially of, or consisting of theamino acid sequence of GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), and/oran isolated peptide comprising, consisting essentially of or consistingof the amino acid sequence of SPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3)and a composition comprising any of these isolated peptides, singly orin any combination in a pharmaceutically acceptable carrier.

Also provided herein is an isolated peptide comprising, consistingessentially of or consisting of about five amino acids to about 15, 20,25, 30, 35, 40, 45, 50, 50, 70, 80, 90 or 100 amino acids (including anyvalue between 5 and 100 not explicitly recited herein), wherein thepeptide comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20) of the 5 mer peptides listed in Table1, as well as a composition comprising any of these isolated peptides,singly or in any combination in a pharmaceutically acceptable carrier.

“Pharmaceutically acceptable,” as used herein, means a material that isnot biologically or otherwise undesirable, i.e., the material may beadministered to a subject along with the compositions of this invention,without causing substantial deleterious biological effects orinteracting in a deleterious manner with any of the other components ofthe composition in which it is contained. The material would naturallybe selected to minimize any degradation of the active ingredient and tominimize any adverse side effects in the subject, as would be well knownto one of skill in the art (see, e.g., Remington's PharmaceuticalScience; latest edition). Exemplary pharmaceutically acceptable carriersfor the compositions of this invention include, but are not limited to,sterile pyrogen-free water and sterile pyrogen-free physiological salinesolution, as well as other carriers suitable for injection into and/ordelivery to a subject of this invention, particularly a human subject,as would be well known in the art.

In some embodiments of this invention, a biomarker peptide and/orprotein of this invention can be used, in any combination, as anantiviral, an antimicrobial, and/or an antifungal agent. Thus, thebiomarker peptides and/or proteins of this invention can be used inmethods of treating and/or preventing disorders such as disordersassociated with fatigue (e.g., chronic fatigue syndrome), viralinfection, disease associated with viral infection, microbial infection,disease associated with microbial infection, fungal infection, diseaseassociated with fungal infection, and any combination thereof. Dosages,modes and regimens of administration for peptides and proteins asdescribed herein would be determined by one of skill in the artaccording to art-known protocols (see, e.g., Remington's PharmaceuticalScience; latest edition).

In some embodiments, the present invention provides a biomarker proteinor peptide of this invention, a nucleic acid comprising a nucleotidesequence encoding a biomarker protein or peptide of this invention, avector comprising said nucleic acid and a cell containing said vector.The biomarker, the nucleic acid, the vector and/or the cell can bepresent singly and/or in any combination in a composition comprising apharmaceutically acceptable carrier.

In other embodiments of this invention, a nucleic acid having thenucleotide sequence or a substantially similar nucleotide sequence ofthe gene encoding a biomarker protein or peptide of this invention canbe used as a probe in a nucleic acid hybridization assay for thedetection of nucleic acid encoding a biomarker protein or peptide invarious tissues and/or body fluids of a subject of this invention. Theprobe can be used in any type of nucleic acid hybridization assayincluding Southern blots (Southern, 1975, J Mol. Biol. 98:508), Northernblots (Thomas et al., 1980, Proc. Natl Acad. Sci. U.S.A. 77:5201-05),colony blots (Grunstein et al., 1975, Proc. Natl Acad. Sci. U.S.A.72:3961-65), slot blots, dot blots, etc. Stringency of hybridization canbe varied depending on the requirements of the assay according tomethods well known in the art. Assays for detecting nucleic acidencoding a protein in a cell, or the amount thereof, typically involvefirst contacting the cells or extracts of the cells containing nucleicacids therefrom with an oligonucleotide probe that specifically binds tonucleic acid encoding a protein or peptide as described herein(typically under conditions that permit access of the oligonucleotide tointracellular material), and then detecting the presence or absence ofbinding of the oligonucleotide probe thereto. Any suitable assay formatcan be employed (see, e.g., U.S. Pat. No. 4,358,535; U.S. Pat. Nos.4,302,204; 4,994,373; 4,486,539; 4,563,419; and 4,868,104, thedisclosures of each of which are incorporated herein by reference intheir entireties).

As used herein, the terms peptide and polypeptide are used to describe achain of amino acids, which correspond to those encoded by a nucleicacid. A peptide usually describes a chain of amino acids of from two toabout 50 amino acids and polypeptide usually describes a chain of aminoacids having more than about 50 amino acids. The term polypeptide canrefer to a linear chain of amino acids or it can refer to a chain ofamino acids, which have been processed and folded into a functionalprotein. It is understood, however, that 50 is an arbitrary number withregard to distinguishing peptides and polypeptides and the terms may beused interchangeably for a chain of amino acids around 50. The peptidesand polypeptides of the present invention can be obtained by isolationand purification of the peptides and polypeptides from cells or bodyfluids or tissues where they are found naturally or by expression of arecombinant and/or synthetic nucleic acid encoding the peptide orpolypeptide. The peptides and polypeptides of this invention can beobtained by chemical synthesis, by proteolytic cleavage of a polypeptideand/or by synthesis from nucleic acid encoding the peptide orpolypeptide.

It is also understood that the peptides and polypeptides of thisinvention may also contain conservative substitutions where a naturallyoccurring amino acid is replaced by one having similar properties andwhich does not alter the function of the peptide or polypeptide. Suchconservative substitutions are well known in the art. Thus, it isunderstood that, where desired, modifications and changes can be made inthe nucleic acid sequence of the underlying gene(s) and/or amino acidsequence of the peptides and polypeptides of the present invention andstill obtain a peptide or polypeptide having like or otherwise desirablecharacteristics. Such changes can occur in natural isolates or can besynthetically introduced using site-specific mutagenesis, the proceduresfor which, such as mismatch polymerase chain reaction (PCR), are wellknown in the art. One of skill in the art will also understand thatpolypeptides and nucleic acids that contain modified and/or syntheticamino acids and nucleotides, respectively (e.g., to increase thehalf-life and/or the therapeutic efficacy of the molecule), as are wellknown in the art, can be used in the methods of the invention.

“Nucleic acid” as used herein refers to single- or double-strandedmolecules which may be DNA, comprised of the nucleotide bases A, T, Cand G, or RNA, comprised of the bases A, U (substitutes for T), C, andG. The nucleic acid may represent a coding strand or its complement.Nucleic acids may be identical in sequence to a sequence that isnaturally occurring or may include alternative codons that encode thesame amino acid as that which is found in the naturally occurringsequence. Furthermore, nucleic acids may include codons that representconservative substitutions of amino acids as are well known in the art.The nucleic acids of this invention can also comprise any nucleotideanalogs and/or derivatives as are well known in the art.

As used herein, the term “isolated nucleic acid” means a nucleic acidseparated or substantially free from at least some of the othercomponents of the naturally occurring organism, for example, the cellstructural components commonly found associated with nucleic acids in acellular environment and/or other nucleic acids. The isolation ofnucleic acids can therefore be accomplished by well-known techniquessuch as cell lysis followed by phenol plus chloroform extraction,followed by ethanol precipitation of the nucleic acids. The nucleicacids of this invention can be isolated from cells according to methodswell known in the art for isolating nucleic acids. Alternatively, thenucleic acids of the present invention can be synthesized according tostandard protocols well described in the literature for synthesizingnucleic acids. Modifications to the nucleic acids of the invention arealso contemplated, provided that the essential structure and function ofthe peptide or polypeptide encoded by the nucleic acid are maintained.

The present invention further provides a kit for detection and/orquantification of the biomarkers of this invention. In some embodiments,such a kit can comprise one or more antibodies, ligands and/or aptamers,along with suitable buffers, wash solutions, dilution buffers, secondaryantibodies, detection reagents, etc., for the detection ofantigen/antibody complex formation, ligand/target complex formationand/or aptamer/target complex formation under various conditions. Inanother embodiment, a kit of this invention can comprise a nucleic acidprobe or primer that is complementary to a nucleotide sequence encodinga biomarker of this invention, along with suitable buffers, washsolutions, dilution buffers, detection reagents, etc. for theamplification of target nucleic acid and/or detection of nucleic acidhybridization under various conditions.

Thus, in some embodiments, the present invention provides a kitcomprising an antibody that specifically reacts with a biomarker of thisinvention and reagents for detecting antigen/antibody complex formation.

Further provided is a kit comprising an aptamer that specifically reactswith a biomarker of this invention and reagents for detectingaptamer/target molecule complex formation.

In addition, a kit is provided herein, comprising a nucleic acid thathybridizes under high stringency conditions with a nucleic acid encodinga biomarker of this invention and reagents for detecting nucleic acidhybridization complex formation.

Screening Methods

In addition, the present invention provides a method of identifying asubstance that binds a peptide or protein of this invention, comprisingcontacting the peptide or protein with a test compound under conditionswhereby binding between the peptide or protein and the test compound canbe detected; and detecting binding between the peptide or protein andthe test compound.

Further provided is a method of identifying a substance having theability to inhibit or enhance the binding activity of a peptide orprotein of this invention, comprising contacting the substance with thepeptide or protein under conditions whereby binding can occur anddetecting a decrease or increase in the amount of binding in thepresence of the substance as compared to a control amount of binding inthe absence of the substance, thereby identifying a substance having theability to inhibit or enhance the binding activity of the peptide orprotein.

For the methods of this invention that employ the detection of binding,such assays are well known in the art and can employ, for example, anantibody, ligand and/or aptamer that binds a peptide of this inventioneither directly or indirectly.

Also provided herein is a method of identifying a compound thatmodulates the activity of a peptide or protein of this invention,comprising contacting the peptide or protein with a test compound underconditions whereby modulation of the activity of the peptide or proteincan be detected. Because there is an association between fatigue and achange in the levels of the peptides and proteins of this invention, thepeptides and proteins may serve a role in, for example, communicating astate of high energy demand to target organs, altering function oforgans involved in mobilization of energy, modulating the activity oforgans involved with the mobilization of energy stores including adiposetissue, the liver and muscle, modulating the activity ofgastrointestinal mucosal leading to increased absorption of sugars,converting amino acids to sugars, or modifying the metabolic andenzymatic activity of commensal bacteria residing in thegastrointestinal tract leading to increased availability of sugars andfree fatty acids that can be used to accomplish physical work byvoluntary muscle, modulating the activity of the liver, pancreas,duodenum and other organs that secrete enzymes, emulsifiers and othersubstances that affect the processing of food, altering the distributionand targeting of sugars, lipids and proteins in the blood. Theseactivities can be measured using in vitro cell-based assays with variousoutput functions that can be used to determine activity, cell-freeassays that measure association with specific receptors or importantregulatory molecules, gene expression assays, and methods that involvemeasurement of functional outputs or alterations of metabolicproduction, fat mobilization and other phenomenon associated withfatigue or the ability to perform physical and cognitive tasks.

Additionally, the present invention provides a method of identifyingimmunomodulating activity in a peptide or protein of this invention,specifically by employing the peptide or protein in an assay forimmunomodulating activity and detecting immunomodulating activity in thepresence of the peptide or protein as compared to a control, therebyidentifying immunomodulating activity in the peptide or protein. In thismethod, the assay for immunomodulating activity can be, but is notlimited to, antibody production (or other assay to detect humoral immuneresponse, T cell activation (or other assay to detect cellular immuneresponse), nitric oxide production, interleukin 2 (IL-2) secretion andany combination thereof.

Furthermore, a method is provided herein of identifying antiviral,antimicrobial and/or antifungal activity in a peptide or protein of thisinvention, comprising employing the peptide or protein in an assay forantiviral antimicrobial and/or antifungal activity and detectingantiviral, antimicrobial and/or antifungal activity in the presence ofthe peptide or protein as compared to a control, thereby identifyingantiviral, antimicrobial and/or antifungal activity in the peptide orprotein. Protocols for identifying antiviral, antimicrobial and/orantifungal activity in a substance are well known in the art.

The term “antibody” as used herein, includes, but is not limited to apolypeptide encoded by an immunoglobulin gene or immunoglobulin genes,or a fragment thereof “Antibody” also includes, but is not limited to, apolypeptide encoded by an immunoglobulin gene or immunoglobulin genes,or a fragment thereof, which specifically binds to and recognizes thebiomarkers of this invention.

The term “epitope” means an antigenic determinant that is specificallybound by an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids and/or sugar side chains and usually havespecific three-dimensional structural characteristics, as well asspecific charge characteristics.

The terms “specifically binds to” and “specifically reactive with” referto a binding reaction that is determinative of the presence of theantigen and antibody or aptamer and target in the presence of aheterogeneous population of proteins, nucleic acids and/or otherbiologics. Thus, under designated assay conditions, the specifiedantibodies and antigens and/or aptamers and targets bind to one anotherand do not bind in a significant amount to other components present in asample.

In some embodiments employing antibodies, a variety of immunoassayformats can be used to select antibodies specifically reactive with aparticular antigen. For example, solid-phase ELISA immunoassays areroutinely used to select monoclonal antibodies specificallyimmunoreactive with an analyte. See Harlow and Lane (ANTIBODIES: ALABORATORY MANUAL, Cold Springs Harbor Publications, New York, (1988))for a description of immunoassay formats and conditions that can be usedto determine specific immunoreactivity. Typically a specific orselective reaction will be at least twice background signal to noise andmore typically more than 10 to 100 times greater than background.

An “immunologically reactive fragment” of a protein refers to a portionof the protein or peptide that is immunologically reactive with abinding partner, e.g., an antibody, which is immunologically reactivewith the protein itself.

Antibodies to biomarkers of this invention can be generated usingmethods that are well known in the art. Such antibodies can include, butare not limited to, polyclonal, monoclonal, chimeric, humanized, fullyhuman, single chain, Fab fragments, and/or fragments produced by anexpression library, including e.g., phage display. (See, e.g., Paul,FUNDAMENTAL IMMUNOLOGY, 3rd Ed., 1993, Raven Press, New York, forantibody structure and terminology.)

Antibody fragments that contain specific binding sites for a biomarkerof this invention can also be generated. For example, such fragmentsinclude, but are not limited to, the F(ab′)₂ fragments that can beproduced by pepsin digestion of the antibody molecule, and the Fabfragments that can be generated by reducing the disulfide bridges of theF(ab′)₂ fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse et al., Science 254,1275-1281 (1989)).

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith a protein or any fragment or oligopeptide or conjugate thereof thathas immunogenic properties. Depending on the host species, variousadjuvants can be used to increase the immunological response. Suchadjuvants include, but are not limited to, Freund's complete andincomplete adjuvant, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Examples of adjuvants used in humans include BCG (bacilliCalmette-Guerin) and Corynebacterium parvum.

Monoclonal antibodies can be prepared using any technique that providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma technique,the human B-cell hybridoma technique, and the EBV-hybridoma technique(Kohler et al. (1975) Nature 256:495-497; Kozbor et al. (1985) J.Immunol. Methods 81:31-42; Cote et al. (1983) Proc. Natl. Acad. Sci.80:2026-2030; Cole et al. (1984) Mol. Cell Biol. 62:109-120). Briefly,the procedure can be as follows: an animal is immunized with a proteinor immunogenic fragment or oligopeptide or conjugate thereof. Lymphoidcells (e.g., splenic lymphocytes) are then obtained from the immunizedanimal and fused with immortalizing cells (e.g., myeloma orheteromyeloma) to produce hybrid cells. The hybrid cells are screened toidentify those that produce the desired antibody.

Human hybridomas that secrete human antibody can be produced by theKohler and Milstein technique and according to art-known protocols.Hybridoma production in rodents, especially mouse, is a very wellestablished procedure and thus, stable murine hybridomas provide anunlimited source of antibody of select characteristics. As analternative to human antibodies, the mouse antibodies can be convertedto chimeric murine/human antibodies by genetic engineering techniques.See Oi et al. Bio Techniques 4(4):214-221 (1986); Sun et al. Hybridoma 5(1986).

The monoclonal antibodies of this invention specific for biomarkerepitopes of this invention can also be used to produce anti-idiotypic(paratope-specific) antibodies. (See e.g., McNamara et al., Science 220,1325-26 (1984); Kennedy et al., Science 232:220 (1986).) Theseantibodies resemble the biomarker epitope and thus can be used as anantigen to stimulate an immune response against the biomarker.

In addition, techniques developed for the production of “chimericantibodies,” the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison et al. Proc. Natl. Acad. Sci.81:6851-6855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takedaet al., Nature 314:452-454 (1985)). Alternatively, techniques describedfor the production of single chain antibodies can be adapted, usingmethods known in the art, to produce biomarker protein-specific singlechain antibodies. Antibodies with related specificity, but of distinctidiotypic composition, can be generated by chain shuffling from randomcombinatorial immunoglobin libraries (Burton, Proc. Natl. Acad. Sci.88:11120-3 (1991)).

Antibodies can also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as described in the literature(Orlandi et al., Proc. Natl. Acad. Sci. 86:3833-3837 (1989)); Winter etal., Nature 349:293-299 (1991)).

Various immunoassays can be used to identify biomarkers of thisinvention. Such immunoassays typically involve the measurement ofantigen/antibody complex formation between a biomarker protein orpeptide and its specific antibody.

The immunoassays of the invention can be either competitive ornoncompetitive and both types of assays are well-known andwell-developed in the art. In competitive binding assays, antigen orantibody competes with a detectably labeled antigen or antibody forspecific binding to a capture site bound to a solid surface. Theconcentration of labeled antigen or antibody bound to the capture agentis inversely proportional to the amount of free antigen or antibodypresent in the sample.

Noncompetitive assays of this invention can be sandwich assays, inwhich, for example, the antigen is bound between two antibodies. One ofthe antibodies is used as a capture agent and is bound to a solidsurface. The other antibody is labeled and is used to measure or detectthe resultant antigen/antibody complex by e.g., visual or instrumentmeans. A number of combinations of antibody and labeled antibody can beused, as are well known in the art. In some embodiments, theantigen/antibody complex can be detected by other proteins capable ofspecifically binding human immunoglobulin constant regions, such asprotein A, protein L or protein G. These proteins are normalconstituents of the cell walls of streptococcal bacteria. They exhibit astrong nonimmunogenic reactivity with immunoglobulin constant regionsfrom a variety of species. (See, e.g., Kronval et al., J. Immunol.,111:1401-1406 (1973); Akerstrom et al., J. Immunol., 135:2589-2542(1985).)

In some embodiments, the non-competitive assays need not be sandwichassays. For instance, the antibodies or antigens in the sample can bebound directly to the solid surface. The presence of antibodies orantigens in the sample can then be detected using labeled antigen orantibody, respectively.

In some embodiments, antibodies and/or proteins can be conjugated orotherwise linked or connected (e.g., covalently or noncovalently) to asolid support (e.g., bead, plate, slide, dish, membrane or well) inaccordance with known techniques. Antibodies can also be conjugated orotherwise linked or connected to detectable groups such as radiolabels(e.g., ³⁵S, ¹²⁵I, ³²P, ¹³H, ¹⁴C, ¹³¹I), enzyme labels (e.g., horseradishperoxidase, alkaline phosphatase), gold beads, chemiluminescence labels,ligands (e.g., biotin) and/or fluorescence labels (e.g., fluorescein) inaccordance with known techniques.

A variety of organic and inorganic polymers, both natural and syntheticcan be used as the material for the solid surface. Nonlimiting examplesof polymers include polyethylene, polypropylene, poly(4-methylbutene),polystyrene, polymethacrylate, poly(ethylene terephthalate), rayon,nylon, poly(vinyl butyrate), polyvinylidene difluoride (PVDF),silicones, polyformaldehyde, cellulose, cellulose acetate,nitrocellulose, and the like. Other materials that can be used include,but are not limited to, include paper, glass, ceramic, metal,metalloids, semiconductive materials, cements and the like. In addition,substances that form gels, such as proteins (e.g., gelatins),lipopolysaccharides, silicates, agarose and polyacrylamides can be used.Polymers that form several aqueous phases, such as dextrans,polyalkylene glycols or surfactants, such as phospholipids, long chain(12-24 carbon atoms) alkyl ammonium salts and the like are alsosuitable. Where the solid surface is porous, various pore sizes can beemployed depending upon the nature of the system.

A variety of immunoassay systems can be used, including but not limitedto, radio-immunoassays (RIA), enzyme-linked immunosorbent assays (ELISA)assays, enzyme immunoassays (EIA), “sandwich” assays, gel diffusionprecipitation reactions, immunodiffusion assays, agglutination assays,immunofluorescence assays, fluorescence activated cell sorting (FACS)assays, immunohistochemical assays, protein A immunoassays, protein Gimmunoassays, protein L immunoassays, biotin/avidin assays,biotin/streptavidin assays, immunoelectrophoresis assays,precipitation/flocculation reactions, immunoblots (Western blot;dot/slot blot); immunodiffusion assays; liposome immunoassay,chemiluminescence assays, library screens, expression arrays, etc.,immunoprecipitation, competitive binding assays and immunohistochemicalstaining. These and other assays are described, among other places, inHampton et al. (Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn. (1990)) and Maddox et al. (J. Exp. Med. 158:1211-1216(1993); the entire contents of which are incorporated herein byreference for teachings directed to immunoassays).

The methods of this invention can also be carried out using a variety ofsolid phase systems, such as described in U.S. Pat. No. 5,879,881, aswell as in a dry strip lateral flow system (e.g., a “dipstick” system),such as described, for example, in U.S. Patent Publication No.20030073147, the entire contents of each of which are incorporated byreference herein.

In some embodiments, the biomarker of this invention can be detectedand/or quantified in an assay employing an aptamer, a molecule thatbinds tightly to the biomarker in a manner similar to an antibody, aligand or a small molecule. As used herein, the term “aptamer” includesany nucleic acid molecule or small peptide that specifically recognizesand binds a target molecule (e.g., a target peptide such as a biomarkerof this invention). An “oligonucleotide-based aptamer” is defined as anaptamer made primarily, although not exclusively, from DNA and/or RNAbases. A “peptide-based aptamer” is defined as an aptamer madeprimarily, although not exclusively, from amino acids.

In some embodiments, an aptamer can be a small, usually stabilized,nucleic acid molecule that includes a binding domain for a targetmolecule (e.g., a biomarker of this invention). Oligonucleotide-basedaptamers of this invention are oligonucleotides, or short (typically<100 bp) polymers of either DNA or RNA that have been selected fromrandom pools based on their ability to bind nucleic acid, proteins,small organic compounds, and even entire organisms, usually with highaffinity.

Oligonucleotide-based aptamers are typically developed to bindparticular ligands using a previously described selection techniquereferred to as SELEX (Systematic Evolution of Ligands by ExponentialEnrichment). This technique allows for selection of aptamers both invivo and in vitro. Methods of making aptamers are described in severalpublications, for example, Ellington and Szostak, Nature 346:818 (1990),Tuerk and Gold, Science 249:505 (1990), U.S. Pat. No. 5,582,981, PCTPublication No. WO 00/20040, U.S. Pat. No. 5,270,163, Lorsch andSzostak, Biochemistry, 33:973 (1994), Mannironi et al., Biochemistry36:9726 (1997), Blind, Proc. Nat'l. Acad. Sci. USA 96:3606-3610 (1999),Huizenga and Szostak, Biochemistry, 34:656-665 (1995), PCT PublicationNos. WO 99/54506, WO 99/27133, WO 97/42317 and U.S. Pat. No. 5,756,291.

Generally, in their most basic form, in vitro selection techniques foridentifying oligonucleotide-based aptamers involve first preparing alarge pool of oligonucleotides of the desired length that contain atleast some central region that is randomized or mutagenized. Forinstance, a common oligonucleotide pool for aptamer selection mightcontain a region of 20-100 randomized nucleotides flanked on both endsby a relatively short (15-25 bp) region of nucleotides with definedsequence useful for the binding of PCR primers. The oligonucleotide poolis amplified using standard PCR techniques.

The original oligonucleotide pool is typically made of DNA bases.However, before the selection step, it can be converted to RNA basesusing in vitro transcription methods well known in the art. During theselection step, the oligonucleotide library is allowed to interact withthe target molecule, which is either free in solution or adhered to aphysical surface such as a bead. In either case, the chemicalenvironment of the interaction is typically controlled to simulateconditions anticipated for the final application of the invention, forexample temperature, pH and osmolality matched to physiologicalconditions. When selection occurs in solution, capillary electrophoresisis used to separate bound from unbound oligonucleotides. For selectionmethods that use solid surfaces, bound and unbound oligonucleotide areseparated by several rounds of washing of the surface. Boundoligonucleotide is isolated and amplified using standard PCR techniques.If the library was converted from DNA to RNA before selection, thenreverse transcription must be used prior to PCR amplification. Theamplified oligonucleotide sequences are then put through another roundof the same type of selection. Typically, the selection process requiresa total of three to ten iterative rounds to produce a high-affinityaptamer. In the final step, the amplified DNA is cloned and sequencedusing standard procedures to identify the sequence of theoligonucleotides that are capable of acting as aptamers for the targetmolecule. Once a sequence has been identified for a tightly bindingoligonucleotide-based aptamer, the nucleotide-based aptamer may befurther refined and optimized for binding affinity by performingadditional rounds of selection starting from a pool of oligonucleotidescontaining controlled levels of randomized mutations of the originaloligonucleotide sequence.

In further embodiments, an oligonucleotide-based aptamer can include atleast one modified nucleotide base. The term “modified nucleotide base”encompasses nucleotides with a covalently modified base and/or sugar.For example, modified nucleotides include nucleotides having sugars thatare covalently attached to low molecular weight organic groups otherthan a hydroxyl group at the 3′ position and other than a phosphategroup at the 5′ position. Such modified nucleotides can also include 2′substituted sugars such as 2′-O-methyl; 2′-O-alkyl; 2′-O-allyl;2′-S-alkyl; 2′-S-allyl; 2′-fluoro; 2′-halo; or 2′-azido-ribose,carbocyclic sugar analogues, a-anomeric sugars; epimeric sugars such asarabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, andsedoheptulose.

Modified nucleotides of this invention can include but are not limitedto, alkylated purines and/or pyrimidines; acylated purines and/orpyrimidines; and other heterocycles. These classes of pyrimidines andpurines are known in the art and include, pseudoisocytosine;N4,N4-ethanocytosine; 8-hydroxy-N6-methyladenine; 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil;5-carboxymethylaminomethyl-2-thiouracil; 5-carboxymethylaminomethyluracil; dihydrouracil; inosine; N6-isopentyl-adenine; 1-methyladenine;1-methylpseudouracil; 1-methylguanine; 2,2-dimethylguanine;2-methyladenine; 2-methylguanine; 3-methylcytosine; 5-methylcytosine;N6-methyladenine; 7-methylguanine; 5-methylaminomethyl uracil; 5-methoxyamino methyl-2-thiouracil; β-D-mannosylqueosine;5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl ester;psuedouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil;4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester;uracil 5-oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil;5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil;5-pentyluracil; 5-pentylcytosine; and 2,6-diaminopurine;methylpseudouracil; 1-methylguanine; and 1-methylcytosine.

Oligonucleotide-based aptamers of this invention can be synthesized fromconventional phosphodiester linked nucleotides using standard solid orsolution phase synthesis techniques that are known in the art. Linkagesbetween nucleotides can use alternative linking molecules. For example,linking groups of the formula P(O)S, (thioate); P(S)S, (dithioate);P(O)NR′2; P(O)R′; P(O)OR6; CO; or CONR′2 wherein R is H (or a salt) oralkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotidesthrough —O— or —S—.

In certain embodiments, the present invention can employ monoclonal orpolyclonal nucleotide-based aptamers. A “monoclonal nucleotide-basedaptamer” as used herein includes a single aptamer with a knownnucleotide sequence. A “polyclonal nucleotide-based aptamer” as usedherein includes a population of aptamers with the same or differentnucleotide sequences that all have an affinity for the same targetmolecule.

In other embodiments, an aptamer of this invention can be a recombinantprotein or peptide that has been selected for specific binding to atarget molecule according to methods known in the art (see, e.g.,Hoppe-Seyler, Crnkovic-Mertens et al. 2004). The peptide-based aptamercan be a short peptide domain inserted into a supporting proteinscaffold that enhances both specificity and affinity by conformationallyconstraining the peptide sequence (Colas, Cohen et al. 1996; Cohen,Colas et al. 1998; Buerger, Nagel-Wolfrum et al. 2003). In someembodiments of the present invention employing a peptide-based aptamer,the term “peptide-based aptamer” can be used to designate the peptide inthe scaffold protein while the term “peptide” can refer to the insertedsequence.

In the methods of the present invention employing peptide-basedaptamers, assays similar to the immunoassays described herein can becarried out to detect and/or quantify a biomarker of this invention,whereby a peptide-based aptamer is used in place of an antibody and anaptamer/target molecule complex, rather than an antibody/antigen complexis detected. The immunoassays described herein can also be adapted toemploy an oligonucleotide-based aptamer in place of an antibody, for thedetection of a nucleic acid/target molecule complex. In someembodiments, the immunoassays of this invention can also be modified toemploy both aptamers and antibodies to detect and/or quantify abiomarker of this invention. Modification of any known immunoassay toaccommodate the detection of binding of a nucleotide- or peptide-basedaptamer to a target molecule would be well known to one of ordinaryskill in the art.

As used herein, the term “signaling aptamer” includes aptamers withreporter molecules, such as a fluorescence dye, attached to the aptamerin such a way that upon conformational changes resulting from theinteraction of the aptamer with a target molecule, the reporter moleculeyields a differential signal, such as, for example, a change influorescence intensity. Alternatively, the amount of target moleculepresent may be quantified by the direct binding and retention of afluorescently tagged aptamer on a solid surface or by the binding of afluorescently tagged aptamer that recognizes the aptamer or antibodythat binds specifically to the target molecule, i.e., secondaryfluorescence assay. Examples of signaling aptamers can be found, forexample, in U.S. Pat. No. 6,706,481, the entire contents of which areincorporated by reference herein for the disclosure of aptamers, methodsof making aptamers and/or methods of using aptamers.

The present invention is more particularly described in the Examples setforth below, which are not intended to be limiting of the embodiments ofthis invention.

EXAMPLES Example 1 Discovery of Salivary Biomarkers for Chronic FatigueSyndrome Sample Collection and Liquid Chromatography-Mass Spectrometry(LC-MS) Analysis

Sample Collection

Saliva samples were collected using a cotton swab-based collectionsystem (Salivette, Sarstedt, Newton, N.C.). Samples were stored at −80°C., shipped on dry ice and processed according to the manufacturer'sinstructions.

Protein Content

The level of protein in each saliva sample was quantified using thecolorimetric bicinchoninic assay (BCA). Absorbance measurements (562 nm)and standard solutions were used to construct a calibration curve andlinear regression was used to determine the final protein concentrationfor the unknown sample.

Size-Based Centrifugal Filtration

The supernatant was spun through a 50 kDa molecular-weight cutoff (MWCO)filter (regenerated cellulose, Millipore, Billerica, Mass.) at 3,000 gfor approximately 1 h at 22° C. The resulting filtrate was loaded into a10 kDa MWCO filter (regenerated cellulose, Millipore) and spun for 1 hat 3,000 g at 22° C. The concentration of peptide and protein in theresulting filtrate was then determined using a commercially availablekit for the BCA assay calibrated using bovine serum albumin standards.To remove salts and increase the concentration of peptides, ˜100 μg ofprotein was passed through a commercially available peptide trap(Michrom, Auburn, Calif.). The peptide concentration was againquantified using the BCA assay. The sample volume was then reduced usinga heated centrifugal concentrator (Centrivap, Labconco, Kansas City,Mo.). Concentrated samples were labeled with a mass-specific variant ofacetic anhydride, i.e., acetic anhydride with either methyl protons ormethyl deuterons (Yu et al., 2004). The labeling mixture consisted of a1:250 dilution of mass-specific acetic anhydride prepared in ethanolwith 50 mM triethylammonium bicarbonate. Samples were incubated for 1 hat 37° C. and then concentrated as described above. Finally, twoaliquots (˜2.5 μg each) from the same sample, labeled separately withlight and heavy forms of acetic anhydride, were combined and injectedonto the LC-MS system.

Ion-Trap Mass Spectrometry Detection

The various components of the processed saliva were separated using anultra-high-pressure liquid chromatography (UPLC) system (Acquity,Waters, Milford, Mass.) with the outlet flowing directly into anion-trap mass spectrometer (Bruker, Esquire 3000+, Billerica, Mass.).The UPLC was configured with a reversed-phase column (BEH300 C18, 1.7 μmparticle, 2.1×100 mm, Waters) and the components were eluted from thecolumn by varying the concentration of methanol in the running bufferlinearly over a range from 10 to 35% at a flow rate of 0.3 ml/min. MSscans were collected at 2-5 Hz.

Analysis of LC-MS Data

A custom analysis program was written in LabVIEW (National Instruments,Austin, Tex.) to allow for the objective and automated identification ofpeak pairs within the data set separated by the expected mass-to-charge(m/z) differences appropriate for labeling with acetic anhydride, e.g.,delta m/z of 3 for a singly charged ion that has been labeled with oneacetate group, delta m/z of 6 for a singly charged ion that has beenlabeled with two acetate groups, etc. Because the overall number of ionsidentified in the CFS samples was lower than typical saliva samplesexamined previously, this analysis also included comparison of singleions, i.e., delta m/z of 0. Cluster analysis was used to identify peakpairs common to the group at the beginning and end of the study.

High-Resolution MS Sequencing of Salivary Peptides

To determine the amino acid sequence of ions of interest, fractions (˜1min wide) of eluent near the elution time of the target ion werecollected from LC injections of labeled saliva. Five fractions werepooled for high-resolution mass spectrometric analysis (12T LTQ-FTUltra, ProSight PC).

Statistical Analysis

Statistical analysis was conducted using R (version 2.11.1) (Team 2010).For box plots, the horizontal line represents the median value while theboundaries of the rectangle indicate the range of the middle twoquartiles. The whiskers indicate a distance 1.5× greater than theinterquartile range from the nearest edge of the box. Open circlesindicate points beyond the whiskers.

Results

Saliva samples were collected and analyzed as described above. Theresulting LC-MS runs were analyzed with custom-written software, whichenabled searches for clusters of mass-peak pairs within each group,i.e., searches for ions that were present in the LC-MS data for themajority of members of a particular group. Each LC-MS run was evaluatedusing the custom application and a list of detected mass pairs waswritten to a text file. In a typical LC-MS run, hundreds of thousands tomillions of peaks were detected, of which several thousand wereseparated by one of the expected mass differences. The locations ofthese ion clusters were plotted with retention time serving as thex-coordinate and m/z serving as the y-coordinate.

From this plot of clusters, sites of potential biomarkers wereidentified, i.e., those sites for which a cluster appeared in one group,but not in the other. The coordinates of these sites were recorded andused to quantify the ion intensity at that location for each individualin the study. Data for three CFS-biomarker candidates are shown in FIGS.1-3.

Quantifying Data Collected in an Ion Trap Mass Spectrometer

For the purposes of the study, the following approach for identifyingbiomarkers has been used. After the small-molecular-weight components ofsaliva were separated, the total protein concentration of the sample wasestimated using a standard BCA assay. Using the estimated proteinconcentration, a total of 4 μg of protein was injected for each samplein an attempt to normalize the amount of material injected.

High-Resolution Mass Spectrometry Data

Amino acid sequence data for three peptides were obtained usinghigh-resolution mass spectrometry. The high-resolution analysis returnedthe following sequences for the three peptides using the single-letteramino acid notation: (1) GNPQGPSPQGGNKPQGPPPPPGKPQ [bm_cfs_cand3], (2)PPGKPQGPPPQGGNQPQGPPPPPGKPQ [bm_cfs_cand4], and (3)SPPGKPQGPPQQEGNKPQGPPPPGKPQ [sp_6]. The genes containing the sequences(1-3) are described below.

Genetic Information for Proteins Containing the Amino Acid Sequence ofthe Peptides

The Proline-rich Salivary Proteins (PRPs) constitute up to 70% of thesoluble protein found in human saliva, and homologous proteins have beenreported in non-human primates as well as in other animals, includingrats, mice and hamsters. In humans, PRPs are the products of two genefamilies located on chromosome 12: (i) the HaeIII family, comprising twoalmost identical genes, PRH1 and PRH2, which code for acidic PRPs, and(ii) the BstN1 family, which includes four genes (PRB1, PRB2, PRB3 andPRB4) and codes for basic PRPs. With post-transcriptional andpost-translational processing, these six genes are responsible for atleast thirteen different human protein products. In addition, a numberof allelic forms, representing minor changes in amino acid composition,have also been identified for each of these genes. A variety offunctions have been suggested for PRPs in saliva including protectionagainst bacterial pathogens, regulation of calcium phosphate deposition,and most recently as a protective mechanism against dietary tannins andother phenolic compounds.

The biomarker with the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ isderived from one of the PRB genes, PRB1, as a primary translationproduct containing 392 amino acids. These two PRB genes code for primarytranslation products of 392 amino acids (PRB1) and 416 amino acids(PRB2). Removal of the signal peptide produces Basic SalivaryProline-rich Protein 1 and Basic Salivary Proline-rich Protein 2, andfurther modifications yield several smaller products from each protein.Two of the final products of the PRB1 gene (Basic Salivary Proline-richProtein 1 and Proline-rich Peptide II-2) contain amino acid sequence (1)[bm_cfs_cand3]. The method of release of the peptide into saliva isunclear. A detailed search of well-characterized proteases did notreveal any with enzymatic specificities that would generate this peptidefragment from the larger proteins.

The biomarker peptide with the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ derives from one, or perhaps both, of a pairof the basic proline-rich protein genes, PRB1 and PRB2, which areclosely linked to the PRH genes. These two genes code for primarytranslation products of 392 amino acids (PRB1) and 416 amino acids(PRB2). Removal of the signal peptide produces Basic SalivaryProline-rich Protein 1 and Basic Salivary Proline-rich Protein 2, andfurther modifications yield several smaller products from each protein.Three of the final products of the PRB1 gene (Basic SalivaryProline-rich Protein 1, Proline-rich Peptide II-2 and Basic PeptideIB-6) and three from the PRB2 gene (Basic Salivary Proline-rich Protein2, Basic Proline-rich Peptide IB-7 and Basic Proline-rich Peptide IB-8c)contain amino acid sequence (2) [bm_cfs_cand4]. As with sequence (1), itis as yet unclear whether only one or both of the PRB1 and PRB2 genes isthe source of amino acid sequence (2).

The biomarker peptide with the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ derives from one of the basic proline-richprotein genes, PRB4. The gene codes for a primary translation productsof 247 amino acids. Removal of the signal peptide produces BasicSalivary Proline-rich Protein 4, and further modifications yield severalsmaller products from each protein. Only one of the final products ofthe PRB4 gene (Basic Salivary Proline-rich Protein 4) contains aminoacid sequence (3) [sp_6].

Example 2 Measurement of Biomarker(s)

-   -   1. Collect a sample of saliva from the test subject.        -   a. Collect saliva by having the subject spit directly into a            collection vial or tube.        -   b. Collect saliva using a matrix-mediated approach, such as            the commercially available Salivette system developed by            Salimetrics.    -   2. Prepare sample for injection into liquid chromatography-mass        spectrometry (LC-MS) system.        -   a. For saliva collected as in (la):            -   i. Spin saliva at 4 k g for 45 min at 4° C.            -   ii. Determine protein concentration using bicinchoninic                acid (BCA) assay with bovine serum albumin (BSA) as                standard.            -   iii. Process saliva through molecular-weight-cutoff                filters using 50 kDa and 10 kDa filters, sequentially.            -   iv. Pass final supernatant through peptide trap                (Michrom, C8).            -   v. Determine protein concentration using BCA assay with                BSA as standard.            -   vi. Dry the sample in a heated chamber with reduced                pressure (Centrivap).            -   vii. Reconstitute the sample in water with 0.1% acetic                acid at a concentration of 0.1 μg of protein/μl.            -   viii. Inject a sample containing 4 μg of protein, as                determined by BCA assay, into LC-MS system.        -   b. For saliva collected as in (1b):            -   i. Extract raw saliva from matrix according to                manufacturer's instructions.            -   ii. Follow steps (2. a.ii-vii)    -   3. Run LC-MS analysis using a linear gradient of acidified        (0.1%) water and methanol (95% to 65% water) to elute compounds        from a reversed-phase column.    -   4. Measure the height of the peak for peptides of interest, as        described herein.    -   5. Inject into the LC-MS system standard peptide solutions in at        least three different concentrations using concentration values        falling within the normal range of each peptide for 4 μg human        saliva.    -   6. Create a calibration curve for the peptide standards by        plotting peak height vs. concentration.    -   7. For each peptide of interest, use the standard curve results        in (6) and divide by 4 μg to determine the relative amount of        peptide per μg of total salivary protein, i.e., the biomarker        index for each peptide.

Example 3 Determining a Threshold Ratio

-   -   1. Using the measurement method described in Example 2, measure        the level of peptide in each sample collected from a group of        subjects (e.g., a population) determined not to have chronic        fatigue syndrome (non-CFS subjects). Such a determination can be        made for example as set forth in Reeves et al. (“Prevalence of        chronic fatigue syndrome in metropolitan, urban, and rural        Georgia” Population Health Metrics 5:5 (2007), the entire        contents of which are incorporated by reference herein).    -   2. Determine the median value of the peptide for the population        of non-CFS subjects.    -   3. Determine the standard deviation of the peptide for the        population of non-CFS subjects.    -   4. Set the threshold ratio for identifying a subject as having        chronic fatigue syndrome or having an increased likelihood of        having or developing chronic fatigue syndrome equal to the        following: Two times the standard deviation as determined in (3)        plus the median as determined in (2).

Example 4 Novel Salivary Biomarker Associated with Chronic FatigueSyndrome

Background.

At present, diagnosis of chronic fatigue syndrome (CFS) requires alengthy and expensive period of clinical examination to rule out allother causes of fatigue. Thus, many patients do not receive timelytreatment or are misdiagnosed. Definitive rapid diagnosis is needed toimprove the clinical outcomes for patients suffering from CFS.

Methodology/Principal Findings.

Saliva samples were obtained during a survey of the population of thestate of Georgia (United States). The goal of this study was todetermine incidence of undiagnosed CFS in the general population.Samples of saliva were maintained as a frozen archive until time ofanalysis. From the archive, samples from 46 subjects with CFS and 45samples from normal controls were selected for further analysis. Theionizable components below 5 kDa were identified using liquidchromatography-mass spectrometry (LC-MS). Patterns of ion intensity asfunctions of retention time and mass-to-charge ratio were compared toidentify significant differences between the CFS and control groups. Asingle biomarker candidate for CFS was identified. This biomarkercandidate was approximately three times more abundant in saliva from CFSsubjects than in saliva from control subjects (p<0.001, Wilcoxon'ssigned rank). The sensitivity and specificity of the biomarker candidatewith respect to correctly identifying CFS are 88 and 91%, respectively.The Receiver Operating Characteristic (ROC) Area-Under-the-Curve (AUC)is 0.935 (95% CI 0.864 to 0.976). De novo sequencing by high-resolutionMS revealed that the biomarker candidate was a peptide of molecularweight 2,633 Da. The amino acid sequence of the identified peptide isfound within the sequence of the saliva-specific 42 kDa basicProline-Rich-Protein, PRB4.

Conclusions/Significance.

A salivary peptide identified as a candidate biomarker for CFS mayfacilitate rapid diagnosis of CFS.

Chronic fatigue syndrome is an orphan disease wrapped in mystery. Theetiology of the disease is not clear nor is its diagnosis and prognosis.Even the Fukuda definition, so frequently cited, describes CFS asunexplained fatigue [1]. The absence of clear etiological contributionsis highlighted by the variety of labels for the condition includingimmune dysfunction syndrome, neuroendocrine immune dysfunction syndrome,allergic encephalomyelitis, post viral syndrome and neurasthenia, amongothers. The self-reported symptoms consistent with CFS include intensefatigue with a duration greater than six months, which is not relievedby rest and causes tiredness that impairs performance of dailyactivities. CFS is associated with a wide spectrum of symptoms includingpain, headaches, cognitive disorders, sleep disorders, anxiety,depression and fatigue exacerbated by exercise. The search for effectivetreatments has been hampered by the lack of comprehension of themolecular and cellular basis for the development and progression of CFS.

The intense fatigue in CFS has been a cause of confusion with severalother chronic conditions such as fibromyalgia, irritable bowel syndrome,and temporomandibular joint syndrome. A lengthy clinical evaluation,including a complete and detailed medical history, should be conductedto rule out other causes of fatigue and to characterize fatigue's form,time of onset, durations, triggering factors, relationship with rest andphysical activities. The lack of objective criteria, specific signsand/or tests for the diagnosis of disease has led to an underestimationof CFS prevalence [2]. Although some have estimated that more than800,000 people suffer from CFS in the US [3], causing a loss of $9billion annually just in earnings and productivity [4], CDC hasprojected that only 9-16% of individuals with CFS had been diagnosed[5]. Even when detected, the average time from the beginning of thesymptoms to the diagnosis of the syndrome is around 5 years [6]. Becausethe fatigue associated with CFS is so severe, patients with CFS are 4times more likely to forgo needed healthcare than non-fatigued subjects[7], worsening the overall health outlook beyond the immediate impact ofthe disease itself.

Specific salivary peptides can be used to determine the physical fatiguestatus of athletes and adults during exercise [10]. Specifically theratio of two endogenous peptides declined by approximately 1,000 foldfrom a rested state to a physically fatigued state over a period ofseveral hours. Following rest, the peptide levels recovered. Bothpeptides are derived from a family of saliva specific proteins callProline Rich Proteins (PRPs). These proteins and their associatedpeptides are only found in the saliva. In a separate study, the ratio ofthese peptides, termed the Fatigue Biomarker Index or FBI, was used toinvestigate physical fatigue levels of candidates for US militarySpecial Forces [11]. These studies showed that a single measurement ofthe FBI made prior to the start of a rigorous selection process lasting12 weeks was one of only four variables needed to predict who wouldultimately pass and who would fail. In general, those candidates whofailed, for reasons related to poor physical performance, had lower FBIlevels, and thus higher levels of baseline fatigue than thoseindividuals that passed. This suggests that levels of the FBI mayindicate a physiological state of fatigue that is persistent andultimately affects performance capability over a relatively long periodof time. Taken together these findings suggested that saliva may providean objective means of evaluating chronic fatigue.

In the current study, a comparative proteomic analysis directed at thelow molecular weight, <5 kD, fraction of saliva was conducted. The goalof the study was to identify differences between CFS and controlsubjects. The results demonstrate that there is at least one peptide,derived from the family PRPs, which is significantly elevated in CFSpatients relative to controls. This peptide offers promise as anobjective diagnostic laboratory test for CFS.

Study Subjects

Study saliva samples were obtained from a large cross-sectionalpopulation based study of CFS and chronic un-wellness in Georgia,investigating the prevalence of CFS between September 2004 and July 2005conducted by the Centers for Disease Control and Prevention[7]. Briefly,10,837 households with 21,165 members were contacted initially bytelephone interview. At the end of screening and selection a total of112 participants met established clinical criteria for CFS using thecriteria established by the 1994 international research case definition[1] using validated test instruments as specified by the InternationalCFS Study Group [12] and CDC standards [13]. A total of 147 subjectsidentified as non-fatigued were identified during the Georgia study. Thecontrol subjects were clinically evaluated and saliva samples wereobtained. Saliva samples were collected from these subjects and controlsfor the purpose of determining salivary cortisol levels [14]. In allcases, the sample obtained at 8 AM was used for the purpose of biomarkerdiscovery. A total of 46 and 45 CFS and control saliva samples,respectively, were obtained for the purpose of biomarker discovery (FIG.4). Samples were stored at −80° C. and shipped on dry ice until thawedfor processing.

Processing of Saliva

Saliva samples were thawed at room temperature and spun for 10 minutesto remove particulates. The resulting supernatant was filteredsequentially through 50 kDa and 10 kDa molecular-weight cutoff filters(regenerated cellulose, Millipore, Billerica, Mass.) to produce a lowmolecular weight fraction of saliva. Approximately 100 ug of peptide, asdetermined by BCA, was desalted using a C-8 column designed for thispurpose (Michrom, Auburn, Calif.).

Ion-Trap Mass Spectrometry to Identify Putative Biomarkers of CFS

Ion-trap mass spectrometers are capable of detecting components betweena m/z of 150 to 2,000 with a precision of 0.1 m/z. The processedlow-molecular weight saliva fraction was introduced onto anultra-high-pressure liquid chromatography (UPLC) system (Acquity,Waters, Milford, Mass.) with the outlet flowing directly into anion-trap mass spectrometer (Bruker, Esquire 3000+, Billerica, Mass.).The UPLC was configured with a reversed-phase column (BEH300 C18, 1.7 μmparticle, 2.1×100 mm, Waters) and the components were eluted from thecolumn by varying the concentration of methanol in the running bufferlinearly over a range from 10 to 35% at a flow rate of 0.3 ml/min. MSscans were collected at 2-5 Hz. An analysis program (PeakQuest, HyperionBiotechnology, San Antonio, Tex.) was used to identify putativebiomarkers. PeakQuest enables identification of changes in the relativeabundance of peptides. In this case, PeakQuest identifies componentpeptides that vary significantly in relative abundance compared tonormal controls.

Definitive Chemical Identification of CFS Biomarkers

All putative biomarkers were found in a normal healthy pool of standardsaliva (Hyperion Biotechnology Inc., San Antonio, Tex.). Portions ofnormal pool saliva were processed in the manner described and fractions1 minute wide corresponding to the elution time were collected.Fractions were combined and concentrated and evaluated usinghigh-resolution mass spectrometry (HRMS) (12T LTQ-FT Ultra, Pro SightPC). HRMS provides a very precise estimation of peptides and peptidefragment mass, e.g., m/z±0.00001. During HRMS, peptide ions arefragmented by electron bombardment leading to specific disruption ofpeptide bonds. The disruption leads to the production of smallerpeptides and individual amino acids obtained from the parent peptide.The mass of these resulting fragments is determined with greatprecision. The estimated masses acquired in this manner are thencompared to theoretical masses that are found on extensive tables. Thetheoretical composition of the fragments enables a reconstruction of theparent peptide. This process is known as de novo sequencing. The de novosequence determined in this manner can be confirmed by synthesizing thepeptide called for from the de novo sequence. If the synthesized peptidedemonstrates similar retention time and m/z on the system used fordiscovery (ion-trap instrument), the identity of the biomarker isconfirmed. The de novo sequence data was used to direct synthesis ofbiomarker peptides. Milligram quantities of biomarkers were synthesized(Anaspec, Fremont, Calif.). The elution times and MS/MS fragmentationpatterns of the synthesized peptides were observed and compared tosimilar data obtained from processed saliva.

Statistical Analysis

Statistical analysis was conducted using SYSTAT (SPSS Inc., Chicago,Ill.). The receiver operator characteristic (ROC) curve was calculatedand analyzed using MedCalc ver. 12.2.1, (MedCalc Software, Mariakerke,Belgium). The standard error associated with ROC associated Area Underthe Curve (AUC) was calculated using the method of DeLong [15].Comparison between control and CFS was made by t-tests. Comparisons ofdistribution of subjects to control and CFS groups according tolifestyle, health factors and demographic factors were made using theChi-square statistic.

A biomarker candidate for CFS was identified. Specifically, levels ofthe biomarker candidate were higher in CFS subjects than control asshown in FIG. 5. Setting a threshold value to 28,840 intensity unitsleads to a test with sensitivity of 91% and specificity of 88%. Withregard to CFS, it is desirable to provide the highest possibleconfidence with regard to diagnosis. To increase confidence in positivediagnosis, a measured value above 35,000 provides 100% confidence of adiagnosis of CFS. However, at this level, 8 of 46 patients that areidentified as having CFS based on clinical signs are incorrectlydetermined to be normal.

Subsequently, de novo sequencing of the putative biomarker using ahigh-resolution mass spectrometer led to chemical identification of thebiomarker candidate, a peptide with the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ. The observed mass of the whole peptide was2725.3907, which is in good agreement with the theoretical mass of2725.3900 (p=6.88 E-73). A peptide of the same sequence was synthesizedand tested on the original LC-MS system used for discovery studies. Theretention time, m/z and fragmentation pattern of the synthesized peptideon the LC-MS system used for discovery were virtually identical to thoseobserved for the ion of interest originally identified in saliva.

The amino acid sequence of the candidate CFS biomarker is found withinthe amino acid sequence of the human salivary protein, Basic ProlineRich Protein 4 (PRB4_HUMAN, P10163). This sequence is not found withinany other known animal, bacterial or plant proteins. In humans, PBR4 isknown to be expressed only in the parotid and other saliva glands.

Analysis was performed to determine if levels of the biomarker candidatewere dependent on various demographic factors. Table 3 shows thedistribution of subjects according to use of medication, gender, race,smoking, regularity of menstruation, menopause status, and obesity.Table 3 indicates that the CFS subjects are more likely to be takingmedication, be smokers and have irregular menstruation than the normalsubjects group. Table 3 shows that age and BMI are not statisticallydifferent between CFS and normal subjects.

Because the distribution of demographic factors is quite differentbetween CFS and normal subjects, it may be argued that the CFS biomarkercandidate identified in this study is instead a measure of factorsrelated specifically to sampling differences. In other words, thebiomarker candidate may be associated with medication usage, smoking orother demographic factors. To examine this possibility, CFS subjectswere compared statistically using t-test. These results are shown inTable 4. Medication status, gender, race, smoking status, quality ofmenstruation, menopause status, and obesity were not associated with CFSbiomarker level. Similarly, relationship of age and BMI to biomarkerlevel was also examined. In this case no association was observedbetween these factors and level of the biomarker in CFS subjects. Takentogether these observations suggest that the CFS biomarker is associatedonly with CFS and not with other factors examined here.

The diagnostic utility was evaluated through construction of a ReceiverOperating Characteristic (ROC) curve. The ROC curve is shown in FIG. 6.The calculated Area-Under-the-Curve (AUC) of this ROC curve is 0.935with a standard error of 0.0319 and with a 95% confidence interval usingthe binomial exact method of 0.864 to 0.976. A diagnostic test that hasROC AUC of 1.0 indicates a test that is able to perfectly discriminatebetween subjects with the disease and those that do not have thedisease. A ROC AUC of 0.5 indicates a test that does no better thanrandom assignment of individuals to groups.

The present study confirms that saliva can be used for detection andevaluation of disease-associated biomarkers and that CFS modulates theexpression of a very specific set of molecules. A battery of biochemicaland bioinformatic tools was employed to identify a 2.7 kDa peptidepresent in the salivary samples from CFS patients, at levels much higherthan that of normal subjects. By identifying the amino acid sequence ofthis peptide and performing a search against the non-redundant GenBank®database, it was determined that the 2.7 kDa peptide is a fragment ofhuman salivary proline-rich protein 4 (PRB4).

PRB4 belongs to the family of human salivary proline-rich proteins(PRPs), which include six closely linked genes on chromosome 12p13.2.All of the PRP genes are similar in structure, with complexelectrophoretic patterns. Each PRP gene is approximately 4.0 kb inlength and contains four exons, the third of which is entirely composedof 63-bp tandem repeats and encodes the proline-rich portion of theprotein products. Exon 3 contains different numbers of tandem repeats inthe different PRP genes. Variation in the numbers of these repeats isalso responsible for length variations in different alleles of the PRBgenes.

Currently, there are no specific biological or morphological biomarkersto establish per se the diagnosis of CFS. Its diagnosis is indefinite,and established through the exclusion of other diseases causing fatigue.Several studies have been conducted toward discovery of CFSbiomarker(s), but the outcomes have been uncertain.

In conclusion, in the present study, a specific salivary biomarker wasdetected in subjects with CFS. Further studies are being designed toevaluate if the identified salivary 2.7 kDa peptide is not only adiagnostic biomarker but also a prognostic tool as well. In addition,other roles for this peptide, e.g., as a potential mediator of diseasedevelopment and its progression, are currently being assessed.

The above examples clearly illustrate the advantages of the invention.Although the present invention has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

Throughout this application, various patents, patent publications andnon-patent publications are referenced. The disclosures of thesepatents, patent publications and non-patent publications in theirentireties are incorporated by reference herein into this application inorder to more fully describe the state of the art to which thisinvention pertains.

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Basic Proline-rich Protein 2 (PRB2; UniProt: P02812)MLLILLSVALLALSSAQNLNEDVSQEESPSLIAGNPQGAPPQGGNKPQGPPSPPGKPQGPPPQGGNQPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPPQGDKSRSPRSPPGKPQGPPPQGGNQPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPPQGDNKSRSSRSPPGKPQGPPPQGGNQPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPPQGDNKSQSARSPPGKPQGPPPQGGNQPQGPPPPPGKPQGPPPQGGNKSQGPPPPGKPQGPPPQGGSKSRSSRSPPGKPQGPPPQGGNQPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPPQGGSKSRSARSPPGKPQGPPQQEGNNPQGPPPPAGGNPQQPQAPPAGQPQ GPPRPPQGGRPSRPPQ5-mer peptides of P02812 MLLIL SPPGK PQGDK PPPGK PPQGG PPPPP LLILL PPGKPQGDKS PPGKP PQGGN PPPPG LILLS PGKPQ GDKSR PGKPQ QGGNK PPPGK ILLSV GKPQGDKSRS GKPQG GGNKP PPGKP LLSVA KPQGP KSRSP KPQGP GNKPQ PGKPQ LSVAL PQGPPSRSPR PQGPP NKPQG GKPQG SVALL QGPPP RSPRS QGPPP KPQGP KPQGP VALLA GPPPQSPRSP GPPPQ PQGPP PQGPP ALLAL PPPQG PRSPP PPPQG QGPPP QGPPP LLALS PPQGGRSPPG PPQGD GPPPP GPPPQ LALSS PQGGN SPPGK PQGDN PPPPG PPPQG ALSSA QGGNQPPGKP QGDNK PPPGK PPQGG LSSAQ GGNQP PGKPQ GDNKS PPGKP PQGGN SSAQN GNQPQGKPQG DNKSR PGKPQ QGGNK SAQNL NQPQG KPQGP NKSRS GKPQG GGNKS AQNLN QPQGPPQGPP KSRSS KPQGP GNKSQ QNLNE PQGPP QGPPP SRSSR PQGPP NKSQG NLNED QGPPPGPPPQ RSSRS QGPPP KSQGP LNEDV GPPPP PPPQG SSRSP GPPPQ SQGPP NEDVS PPPPPPPQGG SRSPP PPPQG QGPPP EDVSQ PPPPG PQGGN RSPPG PPQGD GPPPP DVSQE PPPGKQGGNQ SPPGK PQGDN PPPPG VSQEE PPGKP GGNQP PPGKP QGDNK PPPGK SQEES PGKPQGNQPQ PGKPQ GDNKS PPGKP QEESP GKPQG NQPQG GKPQG DNKSQ PGKPQ EESPS KPQGPQPQGP KPQGP NKSQS GKPQG ESPSL PQGPP PQGPP PQGPP KSQSA KPQGP SPSLI QGPPPQGPPP QGPPP SQSAR PQGPP PSLIA GPPPQ GPPPP GPPPQ QSARS QGPPP SLIAG PPPQGPPPPP PPPQG SARSP GPPPQ LIAGN PPQGG PPPPG PPQGG ARSPP PPPQG IAGNP PQGGNPPPGK PQGGN RSPPG PPQGG AGNPQ QGGNK PPGKP QGGNQ SPPGK PQGGS GNPQG GGNKPPGKPQ GGNQP PPGKP QGGSK NPQGA GNKPQ GKPQG GNQPQ PGKPQ GGSKS PQGAP NKPQGKPQGP NQPQG GKPQG GSKSR QGAPP KPQGP PQGPP QPQGP KPQGP SKSRS GAPPQ PQGPPQGPPP PQGPP PQGPP KSRSS APPQG QGPPP GPPPQ QGPPP QGPPP SRSSR PPQGG GPPPPPPPQG GPPPP GPPPQ RSSRS PQGGN PPPPG PPQGG PPPPP PPPQG SSRSP QGGNK PPPGKPQGGN PPPPG PPQGG SRSPP GGNKP PPGKP QGGNK PPPGK PQGGN RSPPG GNKPQ PGKPQGGNKP PPGKP QGGNQ SPPGK NKPQG GKPQG GNKPQ PGKPQ GGNQP PPGKP KPQGP KPQGPNKPQG GKPQG GNQPQ PGKPQ PQGPP PQGPP KPQGP KPQGP NQPQG GKPQG QGPPS QGPPPPQGPP PQGPP QPQGP KPQGP GPPSP GPPPQ QGPPP QGPPP PQGPP PQGPP PPSPP PPPQGGPPPP GPPPQ QGPPP QGPPP PSPPG PPQGD PPPPG PPPQG GPPPP GPPPQ PPPQG SARSPGRPSR PPQGG ARSPP RPSRP PQGGN RSPPG PSRPP QGGNQ SPPGK SRPPQ GGNQP PPGKPGNQPQ PGKPQ NQPQG GKPQG QPQGP KPQGP PQGPP PQGPP QGPPP QGPPQ GPPPP GPPQQPPPPP PPQQE PPPPG PQQEG PPPGK QQEGN PPGKP QEGNN PGKPQ EGNNP GKPQG GNNPQKPQGP NNPQG PQGPP NPQGP QGPPP PQGPP GPPPQ QGPPP PPPQG GPPPP PPQGG PPPPAPQGGN PPPAG QGGNK PPAGG GGNKP PAGGN GNKPQ AGGNP NKPQG GGNPQ KPQGP GNPQQPQGPP NPQQP QGPPP PQQPQ GPPPP QQPQA PPPPG QPQAP PPPGK PQAPP PPGKP QAPPAPGKPQ APPAG GKPQG PPAGQ KPQGP PAGQP PQGPP AGQPQ QGPPP GQPQG GPPPQ QPQGPPPPQG PQGPP PPQGG QGPPR PQGGS GPPRP QGGSK PPRPP GGSKS PRPPQ GSKSR RPPQGSKSRS PPQGG KSRSA PQGGR SRSAR QGGRP RSARS GGRPS Basic Proline-richProtein 3 (PRB3; UniProt: Q04118)MLLILLSVALLALSSAQSLNEDVSQEESPSVISGKPEGRRPQGGNQPQRTPPPPGKPEGRPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGQPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPHPGKPEGPPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGPPPQGGNQSQGPPPRPGKPEGSPSQGGNKPQGPPPHPGKPQGPPPQEGNKPQRPPPPGRPQGPPPPGGNPQQPLPPPAGKPQGPPPPPQGGRPH RPPQGQPPQ 5-merpeptides of Q04118 MLLIL PPPGK PPQGG GPPPH EGPPP KPQRP LLILL PPGKP PQGGNPPPHP GPPPQ PQRPP LILLS PGKPE QGGNQ PPHPG PPPQG QRPPP ILLSV GKPEG GGNQSPHPGK PPQGG RPPPP LLSVA KPEGR GNQSQ HPGKP PQGGN PPPPG LSVAL PEGRP NQSQGPGKPE QGGNQ PPPGR SVALL EGRPP QSQGP GKPEG GGNQS PPGRP VALLA GRPPQ SQGPPKPEGP GNQSQ PGRPQ ALLAL RPPQG QGPPP PEGPP NQSQG GRPQG LLALS PPQGG GPPPREGPPP QSQGP RPQGP LALSS PQGGN PPPRP GPPPQ SQGPP PQGPP ALSSA QGGNQ PPRPGPPPQG QGPPP QGPPP LSSAQ GGNQS PRPGK PPQGG GPPPR GPPPP SSAQS GNQSQ RPGKPPQGGN PPPRP PPPPG SAQSL NQSQG PGKPE QGGNQ PPRPG PPPGG AQSLN QSQGP GKPEGGGNQS PRPGK PPGGN QSLNE SQGPP KPEGP GNQSQ RPGKP PGGNP SLNED QGPPP PEGPPNQSQG PGKPE GGNPQ LNEDV GPPPR EGPPP QSQGP GKPEG GNPQQ NEDVS PPPRP GPPPQSQGPP KPEGS NPQQP EDVSQ PPRPG PPPQG QGPPP PEGSP PQQPL DVSQE PRPGK PPQGGGPPPR EGSPS QQPLP VSQEE RPGKP PQGGN PPPRP GSPSQ QPLPP SQEES PGKPE QGGNQPPRPG SPSQG PLPPP QEESP GKPEG GGNQS PRPGK PSQGG LPPPA EESPS KPEGP GNQSQRPGKP SQGGN PPPAG ESPSV PEGPP NQSQG PGKPE QGGNK PPAGK SPSVI EGPPP QSQGPGKPEG GGNKP PAGKP PSVIS GPPPQ SQGPP KPEGP GNKPQ AGKPQ SVISG PPPQG QGPPPPEGPP NKPQG GKPQG VISGK PPQGG GPPPR EGPPP KPQGP KPQGP ISGKP PQGGN PPPRPGPPPQ PQGPP PQGPP SGKPE QGGNQ PPRPG PPPQG QGPPP QGPPP GKPEG GGNQS PRPGKPPQGG GPPPH GPPPP KPEGR GNQSQ RPGKP PQGGN PPPHP PPPPP PEGRR NQSQG PGKPEQGGNQ PPHPG PPPPQ EGRRP QSQGP GKPEG GGNQS PHPGK PPPQG GRRPQ SQGPP KPEGPGNQSQ HPGKP PPQGG RRPQG QGPPP PEGPP NQSQG PGKPQ PQGGR RPQGG GPPPR EGPPPQSQGP GKPQG QGGRP PQGGN PPPRP GPPPQ SQGPP KPQGP GGRPH QGGNQ PPRPG PPPQGQGPPP PQGPP GRPHR GGNQP PRPGK PPQGG GPPPR QGPPP RPHRP GNQPQ RPGKP PQGGNPPPRP GPPPQ PHRPP NQPQR PGKPE QGGNQ PPRPG PPPQE HRPPQ QPQRT GKPEG GGNQSPRPGK PPQEG RPPQG PQRTP KPEGQ GNQSQ RPGKP PQEGN PPQGQ QRTPP PEGQP NQSQGPGKPE QEGNK PQGQP RTPPP EGQPP QSQGP GKPEG EGNKP QGQPP TPPPP GQPPQ SQGPPKPEGP GNKPQ GQPPQ PPPPG QPPQG QGPPP PEGPP NKPQR Basic Proline-richProtein 4 (PRB4; UniProt: E9PAL0)MLLILLSVALLALSSAESSSEDVSQEESLFLISGKPEGRRPQGGNQPQRPPPPPGKPQGPPPQGGNQSQGPPPPPGKPEGRPPQGGNQSQGPPPHPGKPERPPPQGGNQSQGTPPPPGKPERPPPQGGNQSHRPPPPPGKPERPPPQGGNQSQGPPPHPGKPEGPPPQEGNKSRSARSPPGKPQGPPQQEGNKPQGPPPPGKPQGPPPAGGNPQQPQAPPAGKPQGPPPPPQGGRPPRPAQGQQPPQ 5-mer peptides of E9PAL0MLLIL PPPGK PPQGG GPPPH GPPPA LLILL PPGKP PQGGN PPPHP PPPAG LILLS PGKPQQGGNQ PPHPG PPAGG ILLSV GKPQG GGNQS PHPGK PAGGN LLSVA KPQGP GNQSQ HPGKPAGGNP LSVAL PQGPP NQSQG PGKPE GGNPQ SVALL QGPPP QSQGT GKPEG GNPQQ VALLAGPPPQ SQGTP KPEGP NPQQP ALLAL PPPQG QGTPP PEGPP PQQPQ LLALS PPQGG GTPPPEGPPP QQPQA LALSS PQGGN TPPPP GPPPQ QPQAP ALSSA QGGNQ PPPPG PPPQE PQAPPLSSAE GGNQS PPPGK PPQEG QAPPA SSAES GNQSQ PPGKP PQEGN APPAG SAESS NQSQGPGKPE QEGNK PPAGK AESSS QSQGP GKPER EGNKS PAGKP ESSSE SQGPP KPERP GNKSRAGKPQ SSSED QGPPP PERPP NKSRS GKPQG SSEDV GPPPP ERPPP KSRSA KPQGP SEDVSPPPPP RPPPQ SRSAR PQGPP EDVSQ PPPPG PPPQG RSARS QGPPP DVSQE PPPGK PPQGGSARSP GPPPP VSQEE PPGKP PQGGN ARSPP PPPPP SQEES PGKPE QGGNQ RSPPG PPPPQQEESL GKPEG GGNQS SPPGK PPPQG EESLF KPEGR GNQSH PPGKP PPQGG ESLFL PEGRPNQSHR PGKPQ PQGGR SLFLI EGRPP QSHRP GKPQG QGGRP LFLIS GRPPQ SHRPP KPQGPGGRPP FLISG RPPQG HRPPP PQGPP GRPPR LISGK PPQGG RPPPP QGPPQ RPPRP ISGKPPQGGN PPPPP GPPQQ PPRPA SGKPE QGGNQ PPPPG PPQQE PRPAQ GKPEG GGNQS PPPGKPQQEG RPAQG KPEGR GNQSQ PPGKP QQEGN PAQGQ PEGRR NQSQG PGKPE QEGNK AQGQQEGRRP QSQGP GKPER EGNKP QGQQP GRRPQ SQGPP KPERP GNKPQ GQQPP RRPQG QGPPPPERPP NKPQG QQPPQ RPQGG GPPPH ERPPP KPQGP PQGGN PPPHP RPPPQ PQGPP QGGNQPPHPG PPPQG QGPPP GGNQP PHPGK PPQGG GPPPP GNQPQ HPGKP PQGGN PPPPG NQPQRPGKPE QGGNQ PPPGK QPQRP GKPER GGNQS PPGKP PQRPP KPERP GNQSQ PGKPQ QRPPPPERPP NQSQG GKPQG RPPPP ERPPP QSQGP KPQGP PPPPP RPPPQ SQGPP PQGPP PPPPGPPPQG QGPPP QGPPP Basic Proline-rich Protein 4 (PRB4; UniProt: P10163)MLLILLSVALLALSSAESSSEDVSQEESLFLISGKPEGRRPQGGNQPQRPPPPPGKPQGPPPQGGNQSQGPPPPPGKPEGRPPQGGNQSQGPPPHPGKPERPPPQGGNQSQGPPPHPGKPESRPPQGGHQSQGPPPTPGKPEGPPPQGGNQSQGTPPPPGKPEGRPPQGGNQSQGPPPHPGKPERPPPQGGNQSHRPPPPPGKPERPPPQGGNQSQGPPPHPGKPEGPPPQEGNKSRSARSPPGKPQGPPQQEGNKPQGPPPPGKPQGPPPPGGNPQQPQAPPAGKPQGPPPPPQGGRPP RPAQGQQPPQ 5-merpeptides of P10163 MLLIL PPPGK PPQGG GTPPP ERPPP KPQGP LLILL PPGKP PQGGNTPPPP RPPPQ PQGPP LILLS PGKPQ QGGNQ PPPPG PPPQG QGPPP ILLSV GKPQG GGNQSPPPGK PPQGG GPPPP LLSVA KPQGP GNQSQ PPGKP PQGGN PPPPG LSVAL PQGPP NQSQGPGKPE QGGNQ PPPGK SVALL QGPPP QSQGP GKPEG GGNQS PPGKP VALLA GPPPQ SQGPPKPEGR GNQSQ PGKPQ ALLAL PPPQG QGPPP PEGRP NQSQG GKPQG LLALS PPQGG GPPPHEGRPP QSQGP KPQGP LALSS PQGGN PPPHP GRPPQ SQGPP PQGPP ALSSA QGGNQ PPHPGRPPQG QGPPP QGPPP LSSAE GGNQS PHPGK PPQGG GPPPH GPPPP SSAES GNQSQ HPGKPPQGGN PPPHP PPPPG SAESS NQSQG PGKPE QGGNQ PPHPG PPPGG AESSS QSQGP GKPESGGNQS PHPGK PPGGN ESSSE SQGPP KPESR GNQSQ HPGKP PGGNP SSSED QGPPP PESRPNQSQG PGKPE GGNPQ SSEDV GPPPP ESRPP QSQGP GKPEG GNPQQ SEDVS PPPPP SRPPQSQGPP KPEGP NPQQP EDVSQ PPPPG RPPQG QGPPP PEGPP PQQPQ DVSQE PPPGK PPQGGGPPPH EGPPP QQPQA VSQEE PPGKP PQGGH PPPHP GPPPQ QPQAP SQEES PGKPE QGGHQPPHPG PPPQE PQAPP QEESL GKPEG GGHQS PHPGK PPQEG QAPPA EESLF KPEGR GHQSQHPGKP PQEGN APPAG ESLFL PEGRP HQSQG PGKPE QEGNK PPAGK SLFLI EGRPP QSQGPGKPER EGNKS PAGKP LFLIS GRPPQ SQGPP KPERP GNKSR AGKPQ FLISG RPPQG QGPPPPERPP NKSRS GKPQG LISGK PPQGG GPPPT ERPPP KSRSA KPQGP ISGKP PQGGN PPPTPRPPPQ SRSAR PQGPP SGKPE QGGNQ PPTPG PPPQG RSARS QGPPP GKPEG GGNQS PTPGKPPQGG SARSP GPPPP KPEGR GNQSQ TPGKP PQGGN ARSPP PPPPP PEGRR NQSQG PGKPEQGGNQ RSPPG PPPPQ EGRRP QSQGP GKPEG GGNQS SPPGK PPPQG GRRPQ SQGPP KPEGPGNQSH PPGKP PPQGG RRPQG QGPPP PEGPP NQSHR PGKPQ PQGGR RPQGG GPPPH EGPPPQSHRP GKPQG QGGRP PQGGN PPPHP GPPPQ SHRPP KPQGP GGRPP QGGNQ PPHPG PPPQGHRPPP PQGPP GRPPR GGNQP PHPGK PPQGG RPPPP QGPPQ RPPRP GNQPQ HPGKP PQGGNPPPPP GPPQQ PPRPA NQPQR PGKPE QGGNQ PPPPG PPQQE PRPAQ QPQRP GKPER GGNQSPPPGK PQQEG RPAQG PQRPP KPERP GNQSQ PPGKP QQEGN PAQGQ QRPPP PERPP NQSQGPGKPE QEGNK AQGQQ RPPPP ERPPP QSQGT GKPER EGNKP QGQQP PPPPP RPPPQ SQGTPKPERP GNKPQ GQQPP PPPPG PPPQG QGTPP PERPP NKPQG QQPPQ Salivary acidicproline-rich phosphoprotein 1/2 (PRH1/PRH2; UniProt: P02810)MLLILLSVALLAFSSAQDLDEDVSQEDVPLVISDGGDSEQFIDEERQGPPLGGQQSQPSAGDGNQDDGPQQGPPQQGGQQQQGPPPPQGKPQGPPQQGGHPPPPQGRPQGPPQQGGHPRPPRGRPQGPPQQGGHQQGPPPPPPGKPQGPP PQGGRPQGPPQGQSPQ5-mer peptides of P02810 MLLIL GGQQS PPQGR GRPQG LLILL GQQSQ PQGRP RPQGPLILLS QQSQP QGRPQ PQGPP ILLSV QSQPS GRPQG QGPPQ LLSVA SQPSA RPQGP GPPQGLSVAL QPSAG PQGPP PPQGQ SVALL PSAGD QGPPQ PQGQS VALLA SAGDG GPPQQ QGQSPALLAF AGDGN PPQQG GQSPQ LLAFS GDGNQ PQQGG LAFSS DGNQD QQGGH AFSSA GNQDDQGGHP FSSAQ NQDDG GGHPR SSAQD QDDGP GHPRP SAQDL DDGPQ HPRPP AQDLD DGPQQPRPPR QDLDE GPQQG RPPRG DLDED PQQGP PPRGR LDEDV QQGPP PRGRP DEDVS QGPPQRGRPQ EDVSQ GPPQQ GRPQG DVSQE PPQQG RPQGP VSQED PQQGG PQGPP SQEDV QQGGQQGPPQ QEDVP QGGQQ GPPQQ EDVPL GGQQQ PPQQG DVPLV GQQSQ PQQGG VPLVI QQQQGQQGGH PLVIS QQQGP QGGHQ LVISD QQGPP GGHQQ VISDG QGPPP GHQQG ISDGG GPPPPHQQGP SDGGD PPPPQ QQGPP DGGDS PPPQG QGPPP GGDSE PPQGK GPPPP GDSEQ PQGKPPPPPP DSEQF QGKPQ PPPPP SEQFI GKPQG PPPPG EQFID KPQGP PPPGK QFIDE PQGPPPPGKP FIDEE QGPPQ PGKPQ IDEER GPPQQ GKPQG DEERQ PPQQG KPQGP EERQG PQQGGPQGPP ERQGP QQGGH QGPPP RQGPP QGGHP GPPPQ QGPPL GGHPP PPPQG GPPLG GHPPPPPQGG PPLGG HPPPP PQGGR PLGGQ PPPPQ QGGRP LGGQQ PPPQG GGRPQ

TABLE 2 Basic Proline-rich Protein 1 (PRB1; UniProt: P04280)MLLILLSVALLALSSAQNLNEDVSQEESPSLIAGNPQGPSPQGGNKPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPPQGDKSRSPRSPPGKPQGPPPQGGNQPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPPQGDKSQSPRSPPGKPQGPPPQGGNQPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPPQGDKSQSPRSPPGKPQGPPPQGGNQPQGPPPPPGKPQGPPQQGGNRPQGPPPPGKPQGPPPQGDKSRSPQSPPGKPQGPPPQGGNQPQGPPPPPGKPQGPPPQGGNKPQGPPPPGKPQGPPAQGGSKSQSARAPPGKPQGPPQQEGNNPQGPPPPAGGNPQQPQAPPAGQPQGPPRPPQGGRPSRPPQ

TABLE 3 Distribution of CFS and normal subjects with regard todemographic and other factors. Expressed as percentage where CFS n = 46and normal subject n = 45. Chi-square statistic. Body weight is definedas normal, BMI between 18.5 to 24.9, overweight 25.0 to 29.9, and obesegreater than 30 (www.cdc.gov/obesity/adult/defining.html). A smoker isdefined as anyone who answered yes to the question “Have you ever smokedcigarettes regularly?” CFS Normal p-value Medication 0.001 Not Medicated43.5 97.8 Medicated 56.5 2.2 Gender 0.059 Female 78.3 69.2 Male 21.730.8 Race 0.385 American Indian 4.3 0.0 Black 15.2 15.6 Other 2.2 0.0White 78.3 84.4 Smoking 0.005 Smoker 67.4 37.8 Non-Smoker 32.6 62.2Menstruation 0.016 Normal 16.7 44.4 Irregular 83.3 55.6 Menopause 0.109Yes 61.1 40.7 No 38.9 59.3 Obesity 0.127 Normal 17.4 33.3 Overweight39.1 40.0 Obese 43.5 26.7 Age 50.4 48.0 0.218 BMI 28.6 27.1 0.100

TABLE 4 Comparison of CFS subjects CFS biomarker level within group CFSSubjects compared Mean SD p-value Medication Not Medicated (n = 20)52160 19727 0.465 Medicated (n = 26) 47736 20737 Gender Female (n = 36)50684 21557 0.43 Male (n = 10) 45969 14632 Race White (n = 36) 4988921060 0.875 Other (n = 10) 48833 17743 Smoking Smoker (n = 31) 4705318888 0.25 Non-Smoker (n = 15) 55047 22387 Menstruation (female only)Irregular (n = 30) 55830 20599 0.525 Normal (n = 6) 49655 21935Menopause (female only) Yes (n = 22) 48307 23014 0.397 No (n = 14) 5442019262 Obesity Normal (n = 8) 52905 20554 0.633 Overweight or Obese (n =38) 48976 20339

What is claimed is:
 1. A method of guiding a human subject's sleepschedule, comprising: a) measuring the concentration of a peptideselected from the group consisting of: 1) a peptide comprising the aminoacid sequence PPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptidecomprising the amino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ IDNO:2), 3) a peptide comprising the amino acid sequenceSPPGKPQGPPQQEGNKPQGPPPPGKPQ (SEQ ID NO:3), and 4) any combinationthereof, in a saliva sample taken from the subject; b) calculating theratio of the concentration of the peptide(s) measured in (a) to thetotal amount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ SEQ ID NO:1]+[GNPQGPSPQGGNKPQGPPPPPGKPQSEQ ID NO: 2]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ SEQ ID NO;3])/total protein(μg); c) having the subject initiate or resume a sleep schedule; d)measuring the concentration of a peptide selected from the groupconsisting of: 1) a peptide comprising the amino acid sequencePPGKPQGPPPQGGNQPQGPPPPPGKPQ (SEQ ID NO:1), 2) a peptide comprising theamino acid sequence GNPQGPSPQGGNKPQGPPPPPGKPQ (SEQ ID NO:2), 3) apeptide comprising the amino acid sequence SPPGKPQGPPQQEGNKPQGPPPPGKPQ(SEQ ID NO:3), and 4) any combination thereof, in a saliva sample takenfrom the subject at one or more time points after (c), wherein thepeptides of (d) are the same as the peptides of (a); e) calculating theratio of the concentration of the peptide(s) measured in (d) to thetotal amount of protein in the sample, according to the equation:([PPGKPQGPPPQGGNQPQGPPPPPGKPQ SEQ ID NO:1]+[GNPQGPSPQGGNKPQGPPPPPGKPQSEQ ID NO: 2]+[SPPGKPQGPPQQEGNKPQGPPPPGKPQ SEQ ID NO;3])/total protein(μg); and f) guiding the subject's sleep schedule by modifying theduration of subsequent sleep periods using the subject's ratio(s) ascalculated in (e), such that an increase in the ratio relative to theprevious ratio leads to a subsequent increase in the duration of thesubject's sleep period, and a decrease in the ratio or a constant ratiorelative to the previous ratio leads to no change in the duration of thesubject's sleep period or a subsequent decrease in the duration of thesubject's sleep period.